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Biological hydrogen production using Citrobacter amalonaticus Y19 to catalyze the water-gas shift reaction Robaire, Sandra 2008

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BiologicalHydrogenProductionusingCitrobacteramalonaticusY19 toCatalyzetheWater-GasShift ReactionbySandra RobaireB.Eng., McGill University,2006A THESIS SUBMITTEDIN PARTIAL FULFILLMENTOFTHE REQUIREMENTSFOR THE DEGREEOFMASTER OF APPLIEDSCIENCEinThe Faculty of GraduateStudies(Chemical andBiological Engineering)THE UNIVERSITYOF BRITISHCOLUMBIA(Vancouver)December 2008© Sandra Robaire, 2008AbstractThis research reports on investigatingCitrobacter amalonaticusY19, achemoheterotrophic facultative bacterium,as a catalyst to produce hydrogenfrom a low-grade synthesis gas stream. Several productionstrategies were considered,with a twostage batch reaction shownto be most effective. This strategybegins with aerobic growthof the organism, where the biomass isgenerated, followed by hydrogenproduction in ananaerobic environment, whereproduction and activationof carbon monoxidedehydrogenase and the CO-inducedhydrogenase enzymes isrequired to catalyzebiological H2 production. The anaerobicenvironment was createdby purging the reactorswith a gas mixture of 40% COin helium (v/v). The ratesof hydrogen production wereanalyzed by measuring the concentrationsof H2, CO, and CO2 during theanaerobic stageby collecting gas samples from the headspace.Samples were also collected fromthefermentation medium to monitorthe concentration of Ciamalonaticus Y]9, organicacids, and pH.Design of experimentswas used to investigate the dependenceof hydrogenproductivity on various processparameters, including reactor pressureand various mediacomponents, such as the presenceof CO during the growthphase, the presence ofglucose, tryptone, and tracemetals such as nickel andiron. The effect of re-suspensionwas also investigated since decouplingthe two stages allows fora larger amount offreedom in selecting processconditions. The results indicate thatincreasing pressure hasa negligible effect on hydrogenproductivity, likely dueto substrate inhibitioncompensating increased substrateavailability. The results alsoshow that altering certainmedia components can increasehydrogen productivity.Nickel, in particular, increasedthe H2 productivity from 0.47to 0.87 mmol H2! (L x h)when its concentration wasincreased from 0 to 125mg!L.Re-suspension between stagesdecreased the inhibitionofenzyme production andactivation by eliminating the inhibitorymetabolites producedduring the growth stage. Thiswas manifested in the formof a reduced lag phase fromapproximately 18 hoursto 5 hours with re-suspension,as well as an increasedH2production rate. The maximumH2 productivity attainedwas 1.5 mmol H2! (L xh) inbuffered media when thecells were re-suspended betweenstages.11Table of contentsAbstract..iiTable of contentsiiiList of tablesvList of figuresviAcknowledgementsx1.0 Introduction11.1 Background11.2 Thesis layout42.0 Literature survey62.1 Hydrogen and fuelcells62.2 Conventional hydrogenproduction methods92.2.1 Steam methane reforming92.2.2 Electrolysis102.3 Biological hydrogenproduction methods122.3.1 Bio-photolysisof water122.3.2 Dark fermentationson carbohydrate-rich substrates142.3.3 Photo-fermentations162.3.4 Water-gas shiftreaction182.4 More on the biologicalwater gas shift reaction202.4.1 Enzymatic pathwayof the bio-WGS202.4.2 Bacterial strainselection for the watergas shift reaction222.4.3 Citrobacter amalonaticusY19253.0 Scope and objectives294.0 Materialsand methods314.1 Materialsand media composition314.1 .1 Microorganism314.1.2 Media composition314.1.3 Design of experiments(DOE)334.2 Methodology334.2.1 Citrobacteramalonaticus Yl 9 growth344.2.2 Hydrogen production344.2.3 Re-suspensionprocedure354.3 Analytical andmeasurement techniques354.3.1 Cell density andpH354.3.2 Gas concentrations364.3.3 Organic acidconcentrations375.0 Results and discussion385.1 Two-stage processfor H2 production fromCO385.1.1 Growthstage385.1.2 Hydrogen productionstage415.2 Effect of COconcentration duringH2 production phase455.3 Effect of mediacomposition505.4 Effect of tracemetals55iii5.5 Strategy for minimizinginhibition of enzyme production615.6 Mass transfer635.7 Effect of cell concentration665.8 Prospects and limitationsof H2 productivity696.0 Conclusions707.0 Recommendations72References74Appendices80Appendix A — Calibration curves80Appendix B — H2 concentrationdata86ivList of tablesTable 1 - Comparison of hydrogenproduction rates by various biohydrogensystems.... 20Table 2 - Summary of properties ofsome bacterial strains used forthe water gas shiftreaction24Table 3 — Lennox’s Luria Bertani mediacomposition (LB)32Table 4 - PTM1 trace salts composition,pre-made from MSL32Table 5 - Metal ion concentrations33Table 6- Analysis of variance (ANOVA) forthe effect of Fe and Ni onH2 productivitybetween 10 and 48 hours57Table 7 - Analysis of variance forthe effect of Ni on H2 productivitybetween 10 and 48hours58VList of figuresFigure 1- How fuel cellswork (Energy informationadministration 2007)2Figure 2 - Fuel cell schematic(Wells et al. 2005)8Figure 3 - Diagram showingthe process of conventionalelectrolysis11Figure 4 - Schematic representationof the WGS pathway.CODH, CO dehydrogenase;Fd, ferrodoxin; ECH, energy-conservinghydrogenase (Henstraet al. 2007) 21Figure 5 - Citrobacteramalonaticus Y19 growthcurve under twoconditions. Symbols:-4-, cell concentrationunder completely aerobicconditions; -s-, cellconcentrationunder sealed aerobicconditions with8% CO (v/v) present in theheadspace.Symbols represent the experimentaldata and lines representthe best-fit modelobtained using CurveExperts1.338Figure 6 - Concentrationof lactate and acetate,as well as pH after thegrowth phase.Hashed boxes, lactate;dotted boxes, acetate.Error bars representthe average of 2measurements40Figure 7— CO, CO2and H2 concentrationsduring the H2 productionphase for a bufferedsystem where growthwas open to air. Purgedwith 40% CO inHe (v/v) at time0.Symbols: -4-,H2;-s-, CO; -A-, CO2. Datapoints represent theaverage of 2 runsand the error bars representthe standard deviation42Figure 8 — VolumetricH2 productivity fora buffered system wheregrowth was open toair. Purged with 40%CO in He (v/v) at time0. Data points representthe average of2 runs and the errorbars represent the standarddeviation43Figure 9 - Batch evolutionof H2 concentrationfor varying initialCO concentrations.COconcentrations vary from7 to 60% CO in Helium (v/v).Symbols: -4-, 7%CO; -m-,14% CO; -A-, 27%CO; -•-, 40% CO;-p-,60% CO. Symbols representtheexperimental data andlines representthe3rd1degree polynomialbest-fit curves46Figure 10 - MaximumH2 productivity andthe time at which themaximum productivitiesare reached for initialCO concentrations rangingfrom 7 to 40%COin He (v/v). ... 47Figure 11 - Effectof the purging gas used(CO or He) on the H2concentration evolution.Initial CO concentrationadjusted after purgefrom 40-100% (v/v).Symbols: -4-,40% CO, purged withHe; -u-, 60% CO,purged with He; -A-,40% CO, purgedwith CO; -•-, 60%CO, purged with CO;--,80% CO, purged withCO; -0-, 100%CO, purged with CO.Symbols represent datapoints, and linesconnect the symbolsfor ease of visualization48Figure 12 - Effect ofaddition of CO duringthe growth phase, additionof glucose in theH2 production phase,and deletion of tryptonein the H2 productionstage on H2productivity comparedto an average productivityfound throughoutthe researchexperiments. Bars representthe average of 2 runsand the error barsrepresent thestandard deviation50Figure 13- Effectof sealing the growthstage with 6.25mLCO in the headspaceunderseveral conditions.Symbols: -+-, LB growth,re-suspended inLB; -s-, SealedLBgrowth with CO; - A-,Sealed LB growthwith CO, buffered;-.-, Sealed LB growthwith CO, re-suspendedin LB. Symbols representaverage of2 experimental runsandvierror bars represent the standard deviation. Lines connect the symbolsfor ease ofvisualization52Figure 14 - Effect of glucose in cell density measured before and after thehydrogenproduction phase. Bars represent the average of 2 runsand the error bars representthe standard deviation54Figure 15 - Maximum H2 productivity for all the combinations of levelsin the factorialdesign on Fe and Ni concentrations. Data bars represent the average of 2or 4 runsdepending on the condition, and the error bars represent the standard deviation. Theaverage time at which the maximum productivity is attained is shown above the databars 56Figure 16 - Effects of nickel and iron on H2 productivity between 10and 48 hours. Graphgenerated by Minitab software during ANOVA analysis57Figure 17 - The effect of nickel concentration on the evolution of112 productivitythroughout the batch H2 production phase when Iron concentrationis kept constantat 32.5mg/L. Symbols: -+-, Omg!L Ni; -•-, 62.5mg/L Ni; -A-, 125mg/LNi. Datapoints represent the average of 4 runs and the error bars represent the standarddeviation59Figure 18 - Effect of nickel on the H2 productivity between 10 and 48 hours,and the timeat which the maximum productivity is reached when Fe is present at the constantlevel of 32.5 mg/L. Data bars represent the average of four measurements and theerror bars represent the standard deviations59Figure 19 - Effect of iron on H2 productivity between 10 and 48 hours, and the timeatwhich the maximum H2 productivity is reached when no Ni is present.Data barsrepresent the average of 4 measurements at 32.5 mg/L and the averageof 2measurements at 125 and 250mg/L. Error bars represent standarddeviation 60Figure 20 - The interaction effect of Ni and Fe on H2 productivity. Graphgenerated byMinitab software during ANOVA analysis61Figure 21 - The effect of re-suspending the cells between the growthand H2 productionphases on H2 concentration evolution. . Symbols: -+-, cells are not re-suspended; -•-, cells are re-suspended between the growth and112 production phases. Datapoints represent the average of 2 runs and the error bars represent thestandarddeviation62Figure 22 - The effect of pressure on H2 concentration. Symbols: -+-, 1atm; -•-, 1.5atm; - A-, 2 atm. Data points represent the average of 2 runs and theerror barsrepresent the standard deviation. The line connects thedata points for ease ofvisualisation64Figure 23 - The effect of pressure on maximum H2 productivity. Databars represent theaverage of 2 runs and the error bars represent the standard deviation65Figure 24 - The effect of quadrupling cell density on112 concentration. Symbols: -4-,regular cell density; -.-, 4 times higher cell density. Data points representtheaverage of 2 runs and the error bars represent the standard deviation67Figure 25 - The effect of quadrupling cell density on maximum hydrogenproductivity.Data bars represent the average of 2 runs and the errorbars represent the standarddeviation67Figure 26 - Hydrogen calibration performed at a pressureof 5psi on May 23’, 2007 .... 80viiFigure 27 - Hydrogen calibration performedat a pressure of 5psi on April22nd2008 ... 81Figure 28 — Carbon monoxide calibration performedat a pressure of 1 ipsi on June29th,200781Figure 29 - Carbon monoxide calibration performed ata pressure of 5psi on November27th,200782Figure 30 - Carbon monoxide calibration performedat a pressure of 5psi on April 23,200882Figure 31 - Carbon dioxide calibration performedat a pressure of 1 ipsi on October 29th,200783Figure 32 - Carbon dioxide calibration performedat a pressure of 5psi on November28th, 200783Figure 33 - Carbon dioxide calibration performed ata pressure of 5psi on April 23rd,200884Figure 34 - Acetate calibration performedon the HPLC on July30th,2008 85Figure 35 - Lactate calibration performed onthe HPLC on July 3 0th, 200885Figure 36- Logarithmic Citrobacter amalonaticusY19 growth curve under twoconditions. Symbols: -+-, cell concentrationunder completely aerobic conditions;-•-, cell concentration under sealed aerobicconditions with 8% CO (v/v)present inthe headspace. Symbols represent data points,and lines connect the symbols for easeof visualization86Figure 37 - Batch evolution of H2 concentrationfor varying initial CO concentrations.CO concentrations vary from 7 to 60% COin helium (v/v). Symbols: -+-, 7% CO; -.-, 14% CO; -A-, 27% CO; -.-, 40%CO;-.-.,60% CO. Symbols represent theexperimental data and lines represent the3’ degree polynomial best-fit curves86Figure 38 - Effect of the purginggas used (CO or He) on the H2 concentrationevolution.Initial CO concentration adjustedafter purge from 40-100% (v/v). Symbols:-4-,40% CO, purged with He; -.-, 60% CO, purgedwith He; -A-, 40% CO, purgedwith CO; -.-, 60% CO. purged withCO; 80% CO. purged with CO; -a-,100%CO, purged with CO. Symbols representdata points, and lines connect the symbolsfor ease of visualization87Figure 39 - Effect of adding 20mM phosphatebuffer to several conditions. Symbols: -4-,LB, no buffer; -.-, LB, buffered; -A-, tryptonedeleted LB, no buffer; -.-, tryptonedeleted LB, buffered; COsealed, no buffer;-(-,CO sealed, buffered. Symbolsrepresent average of 2 experimental runsand error bars represent the standarddeviation. Lines connect the symbols forease of visualization87Figure 40- Effect of Fe concentrationon H2 concentration when no Ni present.Symbols:-4-, 32.5mg/L Fe; -.-, 125mg/LFe; -A-, 250mg/L Fe. Symbols representaverage ofexperimental runs and error bars representthe standard deviation. Lines connect thesymbols for ease of visualization88Figure 41- Effect of Ni concentrationon H2 concentration when 32.5mg/L Fe present.Symbols: -4-, Omg/L Ni; -.-, 62.Smg/LNi; -A-, 125mg/L Ni. Symbols representaverage of experimental runs and error barsrepresent the standard deviation.Linesconnect the symbols for easeof visualization88Figure 42 - Maximum H2 productivity forall the combinations of levels inthe factorialdesign on Fe and Ni concentrations.Data bars represent the averageof 2 or 4 runsdepending on the condition, andthe error bars represent the standard deviation.Theviiiaverage time at which the maximum productivityis attained is shown abovethe databars89Figure 43- Effect of glucose on H2 concentration.Symbols: -+-, No glucose; -•-, Glucosein H2 production phase; - A-, Glucose duringgrowth and H2 production.Symbolsrepresent average of experimental runs anderror bars represent the standarddeviation. Lines connect the symbolsfor ease of visualization89Figure 44- Effect of tryptone deletion, withand without 20mM phosphate buffer.Symbols: -+-, LB, no buffer; -.-, LB, buffered;-A-, Tryptone deleted LB, nobuffer; -.-, Tryptone deleted LB, buffered.Symbols represent averageof 2experimental runs and error bars representthe standard deviation. Lines connectthesymbols for ease of visualization90Figure 45- Effect of sealing thegrowth with 6.25mL CO to the headspaceunder severalconditions. Symbols: -+-, LB growth,re-suspended in LB; -.-, Sealed LBgrowthwith CO; - A-, Sealed LB growth with CO,buffered; -.-, Sealed LB growthwithCO, re-suspended in LB. Symbolsrepresent average of 2 experimentalruns anderror bars represent the standard deviation.Lines connect the symbols for easeofvisualization90Figure 46 - The effect of re-suspendingthe cells between the growthand H2 productionphases on H2 concentration evolution. . Symbols:-+-, cells are not re-suspended;-•-cells are re-suspended between the growthand H2 production phases. Datapointsrepresent the average of 2 runs andthe error bars represent the standarddeviation. 91Figure 47 - The effect of pressureon H2 concentration. Symbols: -+-,1 atm; -u-, 1.5 atm;- A-, 2 atm. Data points representthe average of 2 runs and the errorbars representthe standard deviation91Figure 48- Effect of dilution (decreasedcell density) on H2 concentration.Symbols: -4-,no dilution, 100% cell concentration;-.-, 75% cell concentration; - A-,50% cellconcentration. Points represent theexperimental data and linesconnect the symbolsfor ease of visualization92Figure 49 - The effect of quadruplingcell density on112 concentration. Symbols: -4-,regular cell density; -.-, 4 times highercell density. Data points representtheaverage of 2 runs and the error barsrepresent the standard deviation92Figure 50- H2 concentration profileof maximum conditions. Symbols:-4-, LB; -.-, LB,re-suspended; - A-, LB, re-suspendedand buffered; -.-, LB, re-suspendedand Niadded;..‘,LB, re-suspended, buffered, and Niadded. Symbols represent averageof2 experimental runs anderror bars represent the standarddeviation. Lines connectthe symbols for ease of visualization93ixAcknowledgementsI offer my gratitude to the Natural Sciencesand Engineering ResearchCouncil ofCanada (NSERC) for their funding. Iowe particular thanks to my supervisors,Dr. M.Mohseni and Dr. C. Haynes, who haveprovided answers to myendless questions. Theyhave not only helped guide and structuremy research, but have allowedme to developand plan my project, giving me theopportunity to have significantinput on the focus ofmy research. They have also inspiredme to continue working inthis field of renewableenergy and biofuels.I would also like to thank themany people who have helpedme with myexperiments including Gary Lesnickiand Adriana Cajiao fromthe pilot plant in theMicheal Smith Laboratories(MSL), and Jana Scbmidtova, Dr.Baldwin, and Dr. Creaghfrom the Department of Chemicaland Biological Engineering.The resources madeavailable to me throughtheir laboratories, and the time theyhave taken to teach me themethods to use these resourceshas been paramount tothe effective completionof myexperiments.My fellow students in the Chemicaland Biological EngineeringDepartment atUBC, in particular those frommy lab, were very helpful in keepingme motivatedthroughout my degree. Specialthanks are owed to myparents, who have supportedmethroughout my years of education,both morally and financially.x1.0 Introduction1.1 BackgroundToday’s world energy requirements arestrongly dependant on fossil fuels.Fossilfuels are limited resources and the strongdependence on them is leadingto a fastdepletion of these resources. In 2005, the world’suse of liquid fuels and other petroleumproducts was equivalent to approximately83.6 million barrels oil per day, andthisnumber is growing rapidly and is expectedto exceed 112 million barrels perday by 2030(1 barrel oil equals 42 US gallons or 159liters) (Doman et al. 2008). In additionto thisdepletion, the combustion of fossil fuelsemits large amounts of pollutantslike CO,,NON,SO,, soot, ash, and other organic compounds,which contribute to global climatechanges. In order to limit the amountof emitted pollutants, while still satisfyingtheenergy requirements of the world, a largeamount of research has beendone in the areasof alternative energy sources, chemical feedstocksand efficient processesto maintainsustainable economic growth and reducedependence on petroleum (Amos 2004).While the production of lignocellulosicethanol and other liquidbiofuels isprominent in the market today, hydrogenis sometimes viewed as the next phasein theevolution of renewable fuels becauseof its clean-burning properties.One kilogram ofhydrogen is approximately equivalentto one US gallon of gasoline on an energybasis(Turner et al. 2008). Hydrogen isnot a primary energy source,as hydrogen must beproduced from other substances,but serves as a medium throughwhich primary energysources can be stored, transmittedand utilized to fulfill energyneeds (Das, Veziroglu2001). Fuel cells can use hydrogenand oxygen to make electricity withonly water as aby-product (Figure 1).1Hydrogen Fuel CellInFigure 1- How fuel cells work (Energy informationadministration 2007)Steam methane reforming is the most commonmethod used commerciallytoproduce hydrogen. It uses naturalgas as a feedstock, predominantlycomposed ofmethane, in an energy intensiveand highly endothermie process toproduce a mixture ofhydrogen (H2) and carbon monoxide(CO) called synthesis gas.In order to produceadditional H2, the synthesis gasundergoes high and low temperatureconventional watergas shift reactions with metalcatalysts. Pressure swing adsorptionis then applied toimprove the purity of the H2. The reformingprocess requires high temperatures(>850°C)and pressure (2.5 x106Pa). While H2 production from naturalgas using steam methanereforming is the most common method,many other methods existto produce hydrogenfrom coal, water, and biomass.When biomass is the feedstock,gasification or pyrolysisis the process of choice. For water,electrolysis is the mostcommon method.Biological production of hydrogenis emerging as a viable alternativetechnologyto the energy intensive methods.Not only is biological hydrogenproduction mostlyoperated at ambient temperatures,but it can also utilize biomassincluding wastematerials such as starchand cellulose containing agriculturalor food industry wastes,orcarbohydrate rich industrial wastewaters.Biological112 production pathways include biophotolysis from water,photo-fermentation, darkfermentation, and the watergas shiftreaction. Bio-photolysisuses the photosyntheticpathway in green algaeand2cyanobacteria to split water molecules into hydrogenions and oxygen. Some of thesemicroalgae contain hydrogenase enzymes allowingthe hydrogen ions to be convertedinto hydrogen gas. Manyanaerobic bacteria have also been found to produce hydrogenusing carbohydrate-rich substrates. In the presenceof light, photo-heterotrophic bacteriaconvert organic acids such as acetic, lactic, propionic,and butyric acid into a mixedbiogas composed of H2 and carbon dioxide(C02). Dark fermentations, on the otherhand,do not require light, and produce H2 and CO2 fromcarbon sources such as glucose,sucrose, and more complex starch and cellulosicwastes. Stoichiometric balancessuggestthat the maximum H2 production in dark fermentationis 2 or 4 moles 112 per moleglucose depending on the end product formed during thereaction.While many poorly degradable biomass sourcesand wastes cannot be converteddirectly to hydrogen by microorganisms, gasificationof these feedstocks producessynthesis gas, mainly composed of carbon monoxide(CO). This synthesis gas, along withother CO containing gas streams (e.g. low-gradesyngas used for heating in thedeveloping world), is not an attractive feed streamfor efficient technologies such asconventional WGS reactions. The energyin the waste gas streams can howeverberecovered by producing hydrogen througha biological water-gas shift reaction (bioWGS). The bio-WGS is similar to the conventionalWGS but bacteria are used ratherthan metallic catalysts. When exposedto CO, CO dehydrogenase andhydrogenaseenzymes are induced in several photo-heterotrophicbacteria. The combined activity ofthese two enzymes catalyzes the bio-WGS:CO + H20—H2 + CO2 AG° = -20 kJ mol’(1)Bacterial strains that can undergothe water gas shift include Rhodospirillumrubrum, Rubrivax gelatinosa CBS,Rhodopseudomonas gelatinosa, Rhodopseudomonaspalustris P4, Citrobacter amalonaticus Y]9,and Carboxydothermus hydrogenoformans.Desirable qualities of a bacterial straininclude their ability to grow and producehydrogen with no light, their ability tooperate at a low temperature, andhigh H2productivity. A pure strain is preferableover an undefined mixture forease ofcharacterization. After an extensive reviewof the literature available, itwas found that Ciamalonaticus Y19 is a good candidatebecause it operates at low temperatures,does notrequire light for cell growth or hydrogenproduction, has high hydrogen productionrates3according to the limited publications on this organismto date (Jung et al. 2002), andhasthe ability to grow aerobically, thus faster than organismsthat grow anaerobically.This research has thus focused on investigating Citrobacteramalonaticus Y19, achemoheterotrophic facultative bacterium,as a catalyst to produce hydrogenfrom acarbon monoxide containing gas stream. Twostage batch processes were usedto developa better understanding of the physiologicaland biochemical properties of this organism.The effect of various process parameters, includingpressure and media composition,onhydrogen productivity was investigated over the courseof this research.1.2 Thesis layoutAn overview of the current state of the fieldof hydrogen production with anemphasis on biological hydrogen productionand the water gas shift pathwayis presentedin the extensive literature review in Chapter2. In this chapter, hydrogen andits potentialfor conversion into energy using fuel cellsis introduced in more detail, followedby adescription of conventional hydrogenproduction methods and a reviewof biologicalhydrogen methods. The strain selectionprocess for the water gas shift reaction isthenexplained, along with a more detailedanalysis of the current state ofresearch on theorganism selected.The specific scope and objectivesof the thesis project are presentedin Chapter 3.Chapter 4 details the materials and methodsused throughout the experimentationtoquantify the results. Starting with the originof the bacterial species used, Chapter 4 thendescribes the media used for growthand its composition. This is followedby theprocedure for Ci amalonaticus Y19growth and H2 production, andends with adescription of the analytical and measurementtechniques used throughoutthe researchproject.Chapter 5 outlines the results obtainedusing Ci amalonaticus Y]9to producehydrogen, as well as discussingthe process parameters investigatedand their effects onproductivity. First, the growthphase of Ci amalonaticus Y19is examined with theparameter of CO presence.Then, the H2 production phaseis discussed in depth.Parameters investigated in thisphase include the effect of substrateconcentration and theeffect of media components suchas glucose, tryptone, buffer, andtrace metals like iron4and nickel on H2 productivity and enzymeactivity. Re-suspension of thecells betweenthe growth and hydrogen production stagesis considered as a possiblestrategy forminimizing inhibition. The effect of masstransfer on H2 productivity is assessed andtheprospects and limitations of the H2 productivityobtained are discussed.Finally, Chapter 6 summarizes the resultsfrom this work which serve as a basisfor quantifying the future potentialof this organism as a H2 producer, and Chapter7 givesrecommendations on how to progress andelaborate on the research alreadydone in orderto further develop this process.52.0 Literature surveyThis chapter provides an overviewof the current stateof the field of hydrogenproduction with an emphasison biological hydrogenproduction and the watergas shiftpathway. First, an overviewof why hydrogen is of interestand how it can be usedforenergy purposes is discussed, followedby a description ofconventional hydrogenproduction methods. Biological hydrogenproduction methods arethen highlighted, alongwith their respective microbial species.More details are then givenfor the biologicalwater gas shift reaction includingthe enzymatic pathway andthe process of bacterialstrain selection used for this project.The current state of researchon the organismselected, Citrobacter amalonaticusY19, is then detailed.2.1 Hydrogen andfuelcellsAccording to a report onthe International energy outlookin 2008, worldmarketed energy consumptionis projected to growby 50 percent over the 2005to 2030period. Total world energywill rise from 462 quadrillionBritish thermal units(Btu) in2005 to 563 quadrillion Btuin 2015, and then to695 quadrillion Btu in 2030(Doman etal. 2008). Since oil reservesare finite, there is a need tofocus on an alternativeenergycarrier from a potentially renewablefeedstock. Hydrogen, if producedfrom renewableenergy sources, can be the cleanand sustainable energycarrier of the future.It also hasthe advantage of having anapproximately 3-fold higherenergy content thangasoline(142 MJ/kg for hydrogen versus44.2 MJ/kg for gasoline).Many countries worldwidearecommitted to the possibilityof a hydrogen economy, andorganizations existthat bringthe efforts of thesecountries together suchas the InternationalPartnership for aHydrogen Economy (IPHE)formed in 2003 with 16participating countries.In additionto these organizations,some technologies are alreadyin place such ashydrogen-poweredbuses in Spain, Iceland,and several other countriesacross the world, andhydrogenrefueling stations, includingone at the National ResearchCouncil (NRC) Instituteforfuel cell innovation in Vancouver,BC. In April 2004,the Canadian governmentalsoannounced funding for theCanadian HydrogenHighway to be built betweenVancouverand Whistler, British Columbia.In order to reach apoint where H2 isproduced6commercially worldwide,many advancesneed to be made inthe production, storage,anduses of H2.Hydrogen is themost abundantelement in the universe,constitutingapproximately 75% ofthe universe’s elementalmass (Palmer 2005).However, mostofthe Earth’s hydrogenis in the form ofchemical compoundssuch as waterandhydrocarbons. Elementalhydrogen in the formof diatomic gas,H2, must therefore beproduced from these otherchemical compoundsin order to be usedas an energy carrier.The energy from hydrogencan be released inone of two methods,internalcombustion or the electro-chemicalreaction that occursin fuel cells. Althoughthecombustion process is wellknown as it is similarto the one usedin vehicles today, itdoes not significantlydecrease the productionof nitrous oxidesthat pollute theatmosphere and contributeto climate change. Thenitrous oxidesare created due to thehigh temperaturesgenerated within the combustionchamber, whichcause some of thenitrogen in the airto combine withthe oxygen in the air.Combustionalso releases a lotof energy in the formof heat, which decreasesthe efficiency of theprocess. Fuel cellsarethus the mostpromising energyconversion devicesfor hydrogen and canachieveefficiencies that aretwo to three times greaterthan the internalcombustion engine(Thenational hydrogenassociation a).A fuel cell is a devicethat converts chemicalenergy suppliedas input fuels to thedevice intoelectric energy. Fuelcells are composedof two electrodes,an anode and acathode, and an intermediateelectrolyte layer capableof transferringpositive ions ineither direction, whilea corresponding flowof electrons in an externalcircuit providesthe desired power(Figure 2)(Sorensen2005).In a proton exchangemembrane fuel cell(PEMFC), the intermediateelectrolytelayer is a proton-conductingpolymer membrane.Hydrogen is fedto the anode catalyst(negative electrode)where it dissociatesinto protons andelectrons. The protonsare thenconducted through theelectrolyte membraneto the cathode(positive electrode),but sincethe membrane is electricallyinsulated, the electronstravel throughin an externalcircuitsupplying power.At the cathode catalyst,oxygen reactswith the protonsand theelectrons to form water,which is the onlyby-product of the PEMFC.7bydioxygen(from(air)Anode CathodeFigure 2 - Fuel cell schematic (Wellset al. 2005)Not only are fuel cells pollutionfree, but they are also quietand scalable. Thescalability of fuel cells makesthem ideal for a wide varietyof applications from laptops(50-100 Watts) to centralpower generation (1-200MWatts) (The nationalhydrogenassociation a). Today, fuelcells are still relatively expensiveto build compared tointernal combustion engines;therefore, they will need furtherdevelopment to increasetheir durability and bring downtheir cost in order tocompete economically.One recurring concern thatarises from the idea of usinghydrogen as an energycarrier is that most of the hydrogencurrently being produced isfrom natural gas usingsteam methane reforming,so although PEMFC’s onlyhave water as a by-product,themethane usually comes froma non-renewable source, andsteam methane reformingproduces greenhousegases, which thus does not alleviateglobal warming concerns.Oneanswer to this is thatthe fossil fuels thatare gasified to make hydrogenwould be morecentralized in a smallnumber of generatingplants where the emissionscould bescrubbed, rather than havingair pollution coming frommillions of tail pipes.Anotheranswer is that while inthe near to mid-term, hydrogenwill most likely be producedbysteam methane reforming sinceit is a well understoodand time tested technology,themid- to long-term promisesthat new hydrogen productiontechnologies from renewableresources will becomemore cost effective.2H —82.2 Conventional hydrogen production methodsHydrogen is mainly produced from fossil fuels, water, and biomass. Using fossilfuels, hydrogen can be produced by steam reforming of natural gas, thermal cracking ofnatural gas, partial oxidation of heavy hydrocarbons, and gasification of coal. However,all of these methods are energy intensive processes that require high temperatures(>850°C) (Kapdan, Kargi 2006). Other methods of producing hydrogen include electrolysisor photolysis from water, and pyrolysis or gasification from biomass. While this list is notexhaustive, it shows that there is no shortage of methods that exist for hydrogenproduction.2.2.1 Steam methane reformingCurrently, the least expensive method is steam reforming of natural gas, whichaccounts for nearly 90% of the hydrogen produced industrially (Das, Veziroglu 2001,Crabtree, Dresseihaus & Buchanan 2004). Steam reforming starts from methane, themain constituent of natural gas, which undergoes, along with water vapour, the highlyendothermic reaction,CH4 + H20—*CO + 3H - AH° AG°= +15 lkJ (2)where the enthalpy change z.H° equals 252.3 kJ mor’ or 206.2 kJ mor’ if the input wateris already in gas form. The AH° and G° are reported at ambient temperature andpressure, not at the significantly higher reaction temperature and pressure. This reactionrequires a catalyst, typically nickel or a more complex nickel on aluminum oxide, cobalt,or alkali, and is operated at a high pressure of around 2.5 x106Pa and high temperatureof around 850°C (Sorensen 2005). The reaction is controlled by several factors includingthe reaction temperature, the catalyst, the design of the reactor, and the input steam tomethane ratio. Typical steam to methane ratios are 2 to 3 in order to avoid carbon (char)formation and prevent excess CO formation. High temperatures may also damage thecatalyst and make it possible for methane cracking to occur, producing carbon which mayappear as filaments on the catalyst surface blocking the steam reforming reaction.9In order to produce additional H2, the reformate, also known as synthesis gascomposed mainly of CO and H2, undergoes high and low temperatureconventional watergas shift reactions (WOS) in separate reactors. The WGS is slightly exothermic.CO + H2O—*CO2 + H2 - AH° AG°= -20kJ (3)With the enthalpy change AH° equal to —41.1 kJ mol’ when all reactants are inthe gas form, and —5.0 kJ moE1 if the input water is liquid. The z\H°and AG° are reportedat ambient temperature and pressure. Conceptually, a low temperaturefor the water gasshift reaction would shift the equilibrium to the right favoring theproduction of H2;however, the reaction kinetics are faster at higher temperatures. Thehigh temperaturestage (HTS) can employ a Fe-Cr-based catalyst and is operated at 300-500°Cto reduceCO concentrations to less than 4%. The low temperature stagecan use Cu-Zn-Aloperated at 200-260°C to further reduce the CO concentration to about0.25% (Merida etal. 2004). As these temperatures are lower than the reforming temperature,some heatrecovery can be done by cooling the reactants and recycling theheat to the reformingstage. If the hydrogen produced is required to be very pure, pressureswing adsorption isused to remove the CO2 as well as the un-reacted CO and CH4 from theproduct stream.The main reason for the high cost of productionby steam methane reforming is the heatexchangers needed for the heat recovery between the water gas shift andthe reformer.2.2.2 ElectrolysisWhile steam reforming accounts for about 90%of the H2 in the market, about 5%is attributed to electrolysis of water (Sorensen 2005). Electrolysisof water, whichconsists of splitting hydrogen from water using an electric currentresults in no emissionswhen the electricity is produced via renewable energy.It is a reliable and clean methodthat is capable of producing ultra pureH2 (>99.999%) (Turner et al. 2008), which isnecessary for some applications. Electrolysis is basicallythe reverse fuel cell operationwhere electric energy is converted into hydrogen and oxygen.2H20 - AH°—* 02+ 2H2 (4)At ambient pressure and temperature, the changein enthalpy, AH°, is equal to -288 kJ moE’ for liquid water and —242 kJ moE’ for waterin gas form.10More specifically, the electriccurrent charges the water,breaks the chemicalbondbetween H and 0, and splitsapart the atomic components,creating oppositely chargedions (Figure 3). The negative electrode(anode) attracts thepositively charged hydrogenions, while the positive electrode(cathode) attracts the negativelycharged hydroxideions. As the ions reachthe electrodes, the hydrogenand oxygen gasesrise and arecollected separately.(eH202ftH—beH20Figure 3 - Diagram showingthe process of conventionalelectrolysis(The national hydrogenassociation b)Manufacturers currentlyproduce two types of lowtemperature electrolyzers:alkaline and polymerelectrolyte membraneor proton exchangemembrane (both PEM).The alkaline electrolyzertypically uses an aqueoussolution of waterand 25-30 wt%potassium hydroxide(KOH) as an electrolyte.This liquid electrolyteenables theconduction of ionsbetween the electrodes.Typical alkaline electrolyzersare run withcurrent densities inthe range of l00-300mA/cm2.PEM electrolysis, onthe other hand,uses a solid proton-conductingmembrane thatis not electricallyconductive. Themembrane serves asa gas separation deviceas well as a protonconductor. PEMelectrolyzers are typicallyoperated at higher currentdensities above1 600mA!cm2.WhilePEM electrolyzers requirea higher current density,they avoid the hazardssurroundingKOH and are more easilyable to maintain a significantdifferential pressureacross theanode and cathode (Turneret al. 2008). Currentresearch in this fieldincludes comparingmultiple electrolyzertechnologies (alkalineand PEM) to gaugetheir efficienciesandabilities to be broughton- and off-linequickly, as well as exploringthe synergies of11coproduction of electricityand hydrogen usingwind power. Thisis particularlyinteresting due to the intermittentnature of wind power.According to Kroposki etal. (2006), 39 kilowatt-hours(kWh) of electricityand8.9 liters (L) of water are requiredto produce 1 kilogram(kg) of hydrogen at 25°Cand 1atmosphere pressure. Typicalcommercial electrolyzerssystem efficienciesare 56-73%,which corresponds to 70.1-53.4kWh electricity perkg H2 (Ivy 2004, Kroposkiet al.2006). The cost of electrolysisthus highly dependson the cost of electricity,which is afunction of market fluctuationsand can render thismethod of hydrogenproduction veryexpensive.2.3 Biological hydrogenproduction methodsBiological hydrogen productionprocesses are an alternativeto the energyintensive methods currentlyin place. Not only isbiological hydrogenproduction mostlyoperated at ambient temperaturesand pressures, thus makingit more environmentallyfriendly and inherentlysafer, but this methodof production can also potentiallyutilizerenewable resourcesand in some cases wastematerials such asstarch and cellulosecontaining agriculturalor food industrywastes, or carbohydraterich industrialwastewaters in the formof biomass. In this section,several biologicalpathways used forhydrogen productionwill be describedincluding bio-photolysisof water, darkfermentation, light fermentationand the water gas shiftreaction.2.3.1 Bio-photolysisof waterSome microorganismscan adapt the photosyntheticprocess found inplants andalgae for the productionof hydrogen.2H20—*2H+0AG°=+1498kJ(5)The z\G° is reportedat ambient temperature andpressure. Lightenergy is used inphotosynthesis to splitwater moleculesto hydrogen ions (H)and oxygen. Ingreenplants, the photonsproduced areused only for CO2 reduction;while in somemicroalgae,hydrogenase enzymesare present, allowingfor the hydrogen ionsto be converted intoH2gas. Both eukaryoticgreen algae and prokaryoticcyanobacteria(blue-green algae) have12been shown to produce H2 under certain conditions(Benemann 1997). Bio-photolysisofwater using green algae is termeddirect bio-photolysis while theuse of cyanobacteriainduces indirect bio-photolysis.Bio-photolysis by green algaerequires a period of anaerobicincubation in thedark in order to induce the synthesisand/or activation ofthe hydrogenase enzymes.Green algae that have beenshown to produce hydrogenby bio-photolysis includeChiamydomonas reindardtii (Ghirardiet a!. 2000, Melis 2002)and Scenedesmus obliquus(Florin, Tsokoglou & Happe2001). A study performed byWinkler et al. (Winkler etal.2002) compared the enzyme activityof several algae speciesand found that C.reindhartii had one of the highestactivities at 200nmol/(ig chlorophylla x h). One majorobstacle with bio-photolysisof water is the02 generated which irreversibly inactivatesthe H2 producing system. Usingsulfur-deprived media for culturingC. reindhartii is onepossible solution because itsignificantly decreases the ratesof 02 synthesis and CO2fixation (Ghirardi et a!. 2000).Several studies havebeen done using sulfur-deprivedmedia, and the highest reportedrate ofH2 production forthis system is around 7.95 mmolH2! L after 100 hours (Kosourovet a!. 2002, Melis et al. 2000).This corresponds to 0.07mmol H2! (L x h) in the standardizedunits used by Levin etal. to compare severalbiological hydrogen productionmethods (Levin, Pitt & Love2004).Bio-photolysis by cyanobacteriais termed indirectbio-photolysis becauseitundergoes the followingset of reactions that bothrequire light energy to proceed:12H20+ 6CO2—*C6H12O+ 602 (6)C6H120+ 12H20—÷ 6C0+ 12H2 AG°= +3.2kJ(7)The G° is reported at ambienttemperature and pressure. Hydrogenproductionhas been investigated in many cyanobacterspecies and strains, withinat least 14 genera,and under a wide range of cultureconditions (Pinto, Troshina& Lindblad 2002). Thesespecies may possess severalof the enzymes involvedin H2 synthesis that includenitrogenases, which catalyzethe production of H2 as a by-productof nitrogen reductionto anunonia, uptake hydrogenases,which catalyze the oxidationof H2 synthesized by thenitrogenase, and bi-directionalhydrogenases, whichhave the ability to bothoxidize andsynthesize H2 (Tamagniniet al. 2002). Accordingto a review by Levinet al. (Levin, Pitt& Love 2004), rates of H2 productionby non-nitrogen fixing cyanobacteria,which range13from 0.02 to 0.4 jmolH2! (mg chlorophylla x h), are very lowcompared with thoseofheterocystous cyanobacteria,which rangefrom 0.17 to 4.2 j.imolH2! (mg chlorophylla xh). Anabaena variablilisis responsible forthe high end ofthis H2 productionrange.Anabaena species wereshown to have thehighest hydrogenevolution potential(Pinto,Troshina & Lindblad2002) and thus havebeen the subjectof much research.Accordingto Levin et al., itis a mutant strain of thisspecies, A. variabilisPK84, that has thehighestH2 productivity of 0.355mmol H2!(Lx h) in converted standardizedunits (Sveshnikovetal. 1997).While the H2 productionprocesses usinggreen algae andcyanobacteria canbeconsidered sustainableas they use only wateras a resource andconsume CO2 thusdecreasing air pollutants,their sensitivity to02 along with their low hydrogenproductionpotential make bio-photolysisunattractive as aH2 production method.2.3.2 Dark fermentationson carbohydrate-richsubstratesWhile bio-photolysisrequires a light source,hydrogen canalso be producedin thedark using anaerobicbacteria and carbohydrate-richsubstrates. Productionin the dark isadvantageous becauseof the lower processcost, as photo-bioreactorsare very expensive.These fermentationsproduce a mixedbiogas containing mainlyhydrogen and carbondioxide. Some examplesof anaerobicbacteria that producehydrogen in thismanner arespecies from thegenus Enterobactericeaeand species fromthe genus Clostridium(Junget al. 2002). Dark fermentationscan be operated atmesophilic (25-40°C),thermophilic(40-65°C), and extremethermophilic (65-80°C)temperatures, dependingon the microbebeing used. Hydrogenproductionby these dark fermentationsdepends highlyonenvironmental conditionssuch as pH, temperature,compositionof the substrate,mediacomposition, gaspartial pressure,hydraulic retentiontime, andthe type of microbialculture used (Kapdan,Kargi 2006).Many carbohydrate-richsubstrates can beused for these fermentationsincludingsimple sugars suchas glucose andsucrose, or morecomplex wastessuch as starchcontaining wastes, cellulosecontaining wastes,and food industrywastes or wastewater.Each of thesesubstrates yieldsdifferent amountsof hydrogen,depending onthefermentationpathway and endproducts (Kapdan,Kargi 2006). Catabolismof these14carbohydrate rich substrates first forms pyruvate, which is then metabolized in ananaerobic environment to produce acetyl CoA and either formate or reduced ferredoxin.Cellular ATP is subsequently derived from acetyl-CoA, and H2 is derived from theformate or reduced ferrodoxin (Hallenbeck, Benemann 2002). Enteric bacteria mainly usethe pyruvate-formate lyase enzyme complex to form formate from pyruvate, whilestrictly anaerobic bacteria use pyruvate-ferredoxin oxidoreductase enzyme system toform ferredoxin from pyruvate.Using glucose as a model substrate, stoichiometric balances suggest that whenacetic acid is the end product, the theoretical maximum yield is 4 moles H2 per moleglucose (Equation 8); whereas, when butyric acid is the end product, the theoreticalmaximum yield is 2 moles H2 per mole glucose (Equation 9).C6H120+ 2H20—*2CH3COOH + 2C02+ 4H2 AG°= -1 82.4kJ (8)C6H120—*CH3COOH + 2C02+ 2H2 AG°= -257.lkJ (9)The Gibbs free energy changes are reported at ambient temperature and pressure.If only the H2 yield is considered, thus ignoring the economic value of butyric and aceticacid, acetic acid is the more desirable end product as it results in the higher theoreticalyield; however, the literature suggests that the production of hydrogen does not followthese stoichiometric ratios. Most microbes opt to produce an array of waste products suchas small organic acids and alcohols at the expense of the hydrogen molar yield.Moreover, the production of acids decreases the pH of the medium further lowering theyield of hydrogen (Hallenbeck 2005). While the theoretical maximum hydrogen molaryield is 4 mol H2/mol glucose, current research has been reporting values around 2-3 molH2/mol glucose by using pure cultures or mixed microbial consortiums (Turner et al.2008). While glucose is the most easily fermentable substrate, it is also very costly. Inorder to realize the full potential of fermentation, less expensive and more abundantfeedstocks must be explored. One such abundant and sustainable feedstock islignocellulosic biomass, consisting of hemicellulose (mainly xylose, arabinose andgalactose), cellulose, and lignin. Cellulose is more crystalline than hemicellulose, addingthe difficult challenge of breaking down this polymer into its glucose monomers. In mostcases, using cellulose as a substrate would require an expensive enzymatic break downstep to render the substrate useful.15While dark fermentations using simplesugars have been extensivelycharacterized and give high productionrates, the use of more complex wastematerials isstill in its infancy. Some of themost extensively studied dark fermentationbacteria areClostridium species. Some pure culturesof Clostridium and Enterobacterspecies candegrade insoluble and soluble starch,respectively (Taguchi et al. 1994).OtherClostridium species have been shown to producehydrogen from xylose, xylan, arabinose,galactose, delignified wood fibers, and corn stoverlignocellulose (Taguchi et al. 1996,Levin et al. 2006, Datar et al. 2007).The molar yield for these Clostridiumspeciesconversions was between 2 and 3.2. Clostridiumspecies No.2 has been shown to haveavery high hydrogen production rate of 21 mmolH2!(L x br) using 3% xylose assubstrate(Taguchi et al. 1996). Overall, theH2 synthesis rates vary between 8.2 and 121.0mmolH2!(L x h) (Levin, Pitt & Love 2004).The maximum rate of 121.0 mmolH2!(L x h) wasfound for a mesophilic undefined consortium(mostly Clostridium species) derivedfromdomestic sewage sludge (Chang, Lee& Lin 2002).While much progress has been madeover the past decade in the fieldof hydrogenproduction using dark fermentation pathways,continued research promisesto increase H2yields as well as production rates. Fieldsof particular interest includefinding newbacterial species to convert cellulosicsubstrates directly rather thanneeding an initialenzymatic breakdown step, and metabolicengineering to get aroundthe molar yieldbarrier by redirecting metabolic pathwaysto H2 production rather than by-productpathways such as organic acid production.2.3.3 Photo-fermentationsSome photoheterotrophic bacteria canproduce hydrogen and carbondioxide byconverting organic acids suchas acetic, lactic, propionic, andbutyric acid, while othersproduce hydrogen by convertingcarbon monoxide through thebiological water gas shiftreaction. The latter will bediscussed in section 2.3.4. Whilethe photosystem of purplephotoheterotrophic bacteria isnot powerfiul enough to split water,it can use simpleorganic acids under anaerobicconditions using light energy. Theelectrons liberated fromthe organic carbon are transportedto the electron acceptor ferredoxin.Under nitrogen-16deficient conditions, the nitrogenase enzyme presentin these bacteria reduces protonsinto H2 gas, with the electrons derived from the ferredoxin(Akkerman et a!. 2002).CH3COOH + 2H20 —* 2C0 + 4H2AG°= +lO4kJ (10)The AG° is reported at ambient temperature and pressure.In the presence of N2,the nitrogenase enzyme will catalyze N2 fixationrather than H2 evolution. Thenitrogenase enzyme is also highly sensitive to oxygen,and inhibited by ammonium ionsand high N/C ratios. Since no02 is produced during this process, the sensitivity to02 isnot as critical as during bio-photolysis. Someof the major benefits of photo-fermentations are the high theoretical conversion yields,the lack of02-evolving activity,the ability of the purple photosynthetic bacteriato use a wide spectrum of light, and theability to consume organic substrates derived from wastes,thus giving them a potential tobe coupled with wastewater treatment. The couplingwith wastewater treatment must,nonetheless, first overcome barriers such as reducedlight penetration due to the colour ofthe wastewater (Kapdan, Kargi 2006).Hydrogen production capabilitieshave been investigated for some purplephotosynthetic bacteria such as Rhodobacter spheroides,Rhodobacter capsulatus,Rhodovulurn sulfidophi/urn W-JS,and Rhodospeudornonas palustris (Kapdan,Kargi2006). Hydrogen production rates fromphoto-fermentations vary accordingto manyfactors including light intensity, carbonsource, and the type of microbial cultureused.The highest conversion efficiency fromorganic acids reported in the literaturewas foundusing lactic acid as the carbon source, giving between80 and 86% efficiency (Kapdan,Kargi 2006). The maximum volumetricH2 production rate found was 2.5 mmolH2! (L xh) in converted standardized units using R sphaeroidesRV as a catalyst (Fascetti, Todini1995).Research into photo-fermentations hasalso been done using severalreactorconfigurations including batchprocesses, continuous cultures, andimmobilized cellcultures in or on a solid matrix. Levinet al. reviewed these reactor configurationsandfound that when cells areimmobilized, the rates of hydrogenproduction byphotoheterotrophic bacteria are higher(Levin, Pitt & Love 2004).Although the theoretical yield of photo-fermentationsis quite high, the yields andH2 production rates found sofar are lower than dark fermentationyields. Dark17fermentation produces organicacid by-products thatlower the hydrogenyield onsubstrates such as glucose. Becausephoto-fermentationscan use small organicacids toproduce hydrogen, an interestingnew development andresearch areais the use of anintegrated approach betweendark and photo-fermentations.In this integratedapproach,the organic acids producedby dark fermentationcan be further processedby photo-fermentation to generateadditional hydrogen.This could lead tohigher yields andconversion efficienciesof carbohydrate carbon sourcesinto H2.2.3.4 Water-gasshift reactionThe biological water-gasshift pathway is similarto the conventional watergasshift mentioned in section2.2 (Equation 2), butrather than using a metalliccatalyst, a setof enzymes is utilized. In orderfor this pathway tobe considered, sourcesof CO, whichis the substrate of the reaction,must be available.One source of COis synthesis gas(syngas), which is mainlya mixture of CO,H2, and C02,but mayalso contain minoramounts of methane, N2,and H2S. Current industrialH2 production processesconsistmainly of steam methanereforming which,as mentioned in section2.2, must firstproduce synthesisgas (or syngas) fromcatalytic steamreforming at veryhightemperatures in orderto produce hydrogen. Whilefossil fuels are themost commonsource of syngas, some solidwastes and biomasscan also be gasifiedto produce syngas.Gasification of solid wastesand biomass is morecomplex becauseof the heterogeneityofthe carbon based materials,and not all wastes canbe gasified, but afew promising wastetypes include papermill waste, mixed plasticwaste, forest industrywaste and agriculturalresidues (Sipmaet al. 2006).It is important to makethe distinction betweenlow and high-gradesyngas. Theamount of energyheld in the syngas definesthe grade, whichfor the purposeof thisthesis can be quantifiedin terms of the compositionand temperatureof the syngas. Acombination of a low percentageof CO and H2,as wellas a low temperature,would thusresult in a lower gradesyngas. For quantificationpurposes, and sincethe WGS reactionismostly concerned withthe amount of COavailable, 40% CO(v/v) or higher willbeconsidered high-gradesyngas while 10-20%CO (v/v) will be low-gradesyngas. The cutoff temperature for low-gradesyngas will be assignedat 100°C since temperaturesbelow18this carry less energy. Low-grade syngas, alsoknown as town gas, is abundant indeveloping countries and is used for applications suchas heating but inefficiently. Thislow-grade synthesis gas can be considered a wastestream, and is not an attractivefeedstock option for energy intensive technologiessuch as conventional WGS. Using thislow chemical energy feedstock in the biologicalwater gas shift pathway is thus ofparticular interest to improve the value of this wastestream. Photo-heterotrophic bacteriacan convert low-grade syngas into H2,a useful energycarrier, through bioconversion.The biological water gas shift reaction operatesat ambient temperatures andpressures. Several photo-heterotrophic bacteria canutilize carbon monoxide (CO) as asole carbon source in the presence or absence oflight. When exposed to CO, both acarbon monoxide dehydrogenase (CODH) enzymeand a hydrogenase enzyme areinduced. The combined activity of these two enzymescatalyzes the intracellular water-gas shift reaction:CO + H20—*H2 + CO2 AG°= -2OkJ/mol(11)The AG° is reported at ambient temperatureand pressure. Unlike the hightemperatures used for conventional WGS,the ambient temperatures used for biologicalWGS make CO-oxidation and H2 synthesis thermodynamicallyfavorable, since theequilibrium of this reaction is shifted to the right.Bacterial strains that can undergo thewater gas shift reaction include Rhodospirillumrubrum, Rubrivax gelatinosa CBS,Rhodopseudomonas gelatinosa, Rhodopseudomonaspalustris P4, Citrobacteramalonaticus Y]9, and Carboxydothermushydrogenoformans. More details onthesebacterial strains can be found in section 2.4.Table 1, adapted from Levin et al., summarizesthe maximum production ratesusing the various biological hydrogensystems discussed above (Levin, Pitt &Love2004). The units have been converted into mmolH2! (L x h) in order to make a validcomparison between methods.It is observed that while photolysis and photo-fermentationshave exceedingly lowH2 production rates, the water gas shift reactionis comparable to some dark fermentationresults. The highest production rate isfound using dark fermentation with anundefinedconsortium of mesophilic bacteria, whilethe second highest, at 96 mmol H2/(L xh), isattributed to CO-oxidation by R gelatinosus,using the water gas shift reaction. Usingan19undefined mixture of bacterialstrains may give the highestproduction rates, butscientifically, the results arehard to reproduce and even moredifficult to quantify asthespecific bacteria involved are unlcnown.The water gas shift was,therefore, the pathwaystudied for the remainder of thisthesis because of its great potentialnot only to give highyields of H2,but also to convertwaste syngas to H2.Table 1 - Comparison of hydrogenproduction rates by various biohydrogensystemsH2 synthesis rate H2 synthesisrateB1oH2SystemReferences(reported units) (convertedunits)Direct photolysis4.67 rnmolH2/1180h0.07 mmol H2/(1 x h) (Meliset a!. 2000)Indirect photolysis12.6 nmol H2/jrg protein/h 0.355mmol H2/(1 x h) (Sveshnikov etal. 1997)Photo-fermentation4.0 mlH2/mI/h0.16 mmol H2/(1 x h) (Tsygankovet al. 1998)CO-oxidation0.8 mmol H2/g cdw/min 96.0 mmolH21(l x h) (Woifrumet al. 2002)Dark fermentationsMesophilic, pure strain21.0 mmol H2/l 1/h 21.0nimol H2/(l x h) (Taguchiet al. 1996)Mesophilic, undefmed1,600.0 1H2/m3/h 64.5 mmolH2/(1 x h) (Lay 2000)Mesophilic, undefined3.0 1 H2/l/h 121.0mmol H2/(1 x h) (Changet al. 2002)Thermophilic, undefined198.0 mmolH2/1124 h8.2 mmol H21(l x h)(Ueno et a!. 1996)Extreme thermophilic, pure strain8.4 mmol H2/IJh8.4 mmol H2/(1 x h)(Van Niel et al. 2002)2.4 More on thebiological water gas shfI reaction2.4.1 Enzymaticpathway of the bio-WGSMicroorganisms thatundergo the water gasshift reaction arecalledhydrogenogens. Thesemicroorganisms conserve energyby oxidation of COto CO2coupled to the reductionof protons to H2, catalyzedby CO dehydrogenase (CODH)andhydrogenase, respectively.The energy conserving mechanismused by thesehydrogenogens is stillunknown as it has been foundthat CODH and hydrogenasedo notconserve energy inthe same manneras the classical theories,substrate levelphosphorylation (SLP) and electrontransfer phosphorylation (ETP)(Sipma et al. 2006).Instead, it has been proposedthat a membrane associatedenzyme complex is formedbyCODH and hydrogenase,which facilitates CO oxidationand proton reduction (Figure4).20CO+H202HnHCODH ECHCO2÷2HH2/nHFigure 4 - Schematic representation of the WGS pathway. CODH, CO dehydrogenase;Fd,ferrodoxin; ECH, energy-conserving hydrogenase (Henstra eta!. 2007)The two key enzymes necessary for the biological watergas shift reaction toproceed are CODH and hydrogenase. CODHs arenickel containing,02 sensitiveenzymes, and are either monofunctional or bifunctional(Lindahi 2002). Whilemonofunctional CODHs catalyze the oxidation ofCO as part of the energy metabolism,bifunctional CODHs catalyze the synthesis of acetyl-CoAor its decarbonylation besidethe oxidation of CO (Equation 12).CO + H20-*2H + CO2 + 2& (12)Monofunctional CODHs have been identified inseveral microorganisms that undergo thebiological WGS including 1?. rubrum and C. hydrogenoformans.These enzymes arefunctionally associated with the iron-sulfur proteinCooF, that contains 4 [4Fe-4S]clusters (Siprna et al. 2006).Hydrogenases are the other key enzymes andthey catalyze the reduction ofprotons to H2:2H+2e€-*H2(13)Three classes of hydrogenases exist based on phylogenyand metal content: [NiFe]hydrogenases, [FeFe]-hydrogenase, and iron-sulfurcluster free hydrogenases. Energyconverting hydrogenases (ECHs) are a subclassof [NiFe]-hydrogenases. ECHs aremembrane bound enzyme complexes thatplay a key role in energy generation in thehydrogenogenic metabolism (Hedderich 2004).The overall enzymatic process thus starts with theoxidation of CO by amonofunctional CO dehydrogenase. The electronsthat are released by the oxidation areEd21then transferred to the ECH that reduces theprotons to form molecular112. Since theECH is membrane bound, it couples the formationof H2 to the membrane translocationof protons or sodium ions, creating an ion gradientthat can drive ATP synthesisthroughan ATP-synthase. Energy conservationin these types of microorganismsis thusindependent of the acetyl-CoA pathway (Henstraet al. 2007).2.4.2 Bacterial strain selectionfor the water gas shift reactionThe feasibility of biological H2 productionfrom CO largely depends on theperformance of the microorganism that catalyzesthis reaction, and considerable effortstoisolate appropriate strains have been conductedthus far. The partial listin the literatureincludes several anaerobic photosyntheticbacteria such as Rhodospirillumrubrum, ananaerobic bacterium, Carboxydothermushydrogenoformans, and severalfacultativebacteria, such as Rubrivivax gelatinosusCBS, Citrobacter amalonaticusY19, andRhodospeudomonas palustris P4. The majorityof bacterial strains that catalyzethe WGSare purple non-sulfur photosynthetic bacteria,a non-taxonomic group. These bacteriacangrow as photoheterotrophs, photoautotrophsor chemoheterotrophs, switchingfrom onemode to another depending on the availableconditions such as: degree of anaerobiosis,availability of carbon sources, andavailability of a light source (Basak,Das 2007).R rubrum, a photosynthetic purplenon-sulfur bacterium, has drawnmuchattention due to the high specificCO uptake rate and the high conversionyield close tothe theoretical value (Jung et al. 1999a). However,R rubrum always requires light for itsgrowth and the growth is relativelyslow. In addition, H2 productionis inhibited bymoderate CO partial pressuresabove 0.2 atm.Rhodopseudomonas bacteria arealso purple non-sulfur phototrophicorganisms,and they can be found inmany types of marine environmentsand soils.Rhodopseudomonas palustris P4has a high growth rate of 0.3h’ anda moderate rate ofhydrogen production 20.7 mmol/(g cell x h)(Jung et al. 1999a). While Rh palustrisP4was not inhibited by high CO concentrationsalmost up to 1 atmosphere, thelightrequirement for cell growth wasan important drawback.Carboxydothermus hydrogenoformansis a gram positive, thermophilic,strictanaerobic bacterium thatwas isolated from hydrothermalfreshwater springs in Russia.22This bacterium has the abilityof using CO as the sole carbonsource and has been shownto grow on CO with a doublingtime of 2 hours. While thermophilicorganisms canbeadvantageous because ofhigher conversion rates,higher temperaturesalso have anegative impact on the solubilityof CO in water. Therate of112 production reported inthe literature is 48.7 mmolH2 IL of medium after 45hours (Henstra, Stams2004). It hasalso been shown thatCO thresholds below 2ppmcould be obtained ifCO2 was removedfrom the gas phase of batch culturesusing C hydrogenoformans.Without CO2 removal,lI7ppm CO remained froman original gas phaseof 100% CO, whereas conventionalchemical water gas shift technologyleaves l000ppmof CO (Henstra et al. 2007).Citrobacter is a genusof gram-negative bacteriain the familyof theEnterobacteriaceae.It can be isolated from wastewatersludge digesters.Ci amalonaticusY19 is a facultative anaerobe,able to grow aerobicallyor anaerobically, althoughmuchfaster growth was observedunder aerobic conditions.Hydrogen production,however,was observed only underanaerobic conditions.Since the conditionsfor cell growth andhydrogen production werevery different, a two-stepprocess, which separatesthe growthphase from the hydrogen productionphase, has been suggestedfor maximizing hydrogenproduction. The maximumhydrogen productionactivity found was27.1 mmol H2/(gcellx h) (Jung et al. 2002).Rubrivivax gelatinosusCBS is a non-sulphur, purplephotosynthetic bacteriathatis also capable of performingthe water-gas shift reactionunder anaerobicconditions,atmospheric pressureand an ambient temperatureof 25°C (Amos 2004).Rx GelatinosusCBS can obtain energy inseveral ways includingphotosynthesis,aerobic heterotrophicmetabolism, and anaerobicfermentation pathways.The biological watergas shift reactioncan utilize dark anaerobicfermentation. This pathwayproduces muchless energy thanthe aerobic or photosyntheticpathways, meaninga lower growth rate,but also producesless waste than theother pathways. Indarkness, the CO oxidationpathway containsCOdehydrogenase (CODH)and hydrogenase enzymes,both of whichare induced in CO,similar to that reportedfor R rubrum (Maness,Weaver 2002). Rxgelatinosus CBSwasfound to havea doubling time of approximately7 hours in the light whenCO was used asthe only carbon sourceand the maximumspecific hydrogen productionrate was foundtobe 48 mmol H2!(g cell x h) (Wolfrum, Watt& Huang 2002, Maness,Weaver 2002)23Several selection criteriawere used to determinewhich bacterial strainwould beused for this thesis researchproject. Thefirst criterion is the abilityof the bacterialstrainto grow and producehydrogen with nolight in orderto decrease thecost of the processby eliminating expensivephoto-bioreactors.A second criterionis their abilityto operateat a low temperaturein order to increasethe substrate availabilitysince lowertemperatures resultin higher CO solubilityin water, and highH2 productivity.A purestrain is preferable overan undefined mixturefor ease of characterizationand availabilityof the organism is crucialin order to obtaina sample of theorganism for study.Table 2summarizes many ofthe qualities desiredfor these bacteria,as well as theirmaximum H2production ratesfound in the literature.Table 2 - Summaryof properties of somebacterial strains usedfor the watergas shift reactionCan use CO asTemp Need lightsole energyMax H2 productionReferencerate(°C) for growth?source?30 YesNot specified 11 mmol/(g cellx h)(Klasson et al.Rhodospirillum1992)rubrum0.87 mmol H2 /mmol(Najafpour,30 YesNot specifiedYounesi &Mohamed 2004)Rhodopseudomonas(Jung et al.30 YesNo 20.7 mmol/(gcell x h)palustris P41999a)CitrobacterYes (but use C3 0-40 No27.1 mmol/(g cell xh) (Jung eta!. 2002)amalonaticusY 19source too)70 NoYesNot lcnown(Soboh, Linder &CarboxydothermusHedderich 2002)hydrogenoformansYes (but use 48.7mmol/L medium(Henstra, Stams60-65 Nopyruvate too)(in approx 45 hrs)2004)Rubrivivax25No48 mmol/(g cellx h) (Maness, WeaverYesgelatinosus CBS(but uses it)(96 mmol/ L h)2002)Anaeorobic(Sipma et a!.55 NoYesNot known2004, Sipma eta!.bioreactorsludges2003)All of these bacteriahave their advantagesand disadvantages,and performdifferentlyin terms of hydrogenproduction ratesand growthrates. R rubrumand RhPalustris P4need light for growth,and thus willnot be used forthis study. Oftheremaining strains,Ci amalonaticusY19, C hydrogenoformans,and Rx gelatinosusCBSare pure strains,but although noneof these requirelight for growth,after closer analysis,24it is suggested that Rx gelatinosusCBS prefers light and growsvery slowly in the absenceof it. Between the remainingtwo strains, Ci amalonaticusYl 9 operates at the lowertemperature and thus mayallow for the alleviationof any substrate masstransferlimitations that occur in theprocess. Ci amalonaticusY19 was the bacterial strainselected for further studies.It served as a good systemfor study using the low-pressurereactors available.2.4.3 Citrobacter amalonaticusY19As Ci amalonaticus Y19 was the bacterialstrain used for thisstudy, this sectionwill provide more detailson the strain characteristicsand the parameters studiedin theliterature so far. This organismwas first isolatedfrom an anaerobic wastewatersludgedigester by the researchersat the Pusan national universityin Korea. This research grouphas studied the bacteriaas a catalyst for H2 productionusing both the water gasshiftreaction and dark fermentationpathways. In this review,the focus will be on theuse ofCi amalonaticus Y19 to catalyzethe WGS pathway, whichwas predominantly studiedinbatch serum-bottle cultivations(Jung et aT. 2002, Kim et al.2003, Jung et al. 1999b).An important first discoverywas that Ci amalonaticus Y]9grew fast withaspecific growth rate of0.7 h’ aerobically to a celldensity of 2 g/L, whereasunderanaerobic conditions,the cell specific growthrate and the maximumcell density wereonly 0.12 h’ and 0.4g/L, respectively.It was also shown that no H2 couldbe producedaerobically, which suggeststhat it would be desirableto separate the growth phasefromthe H2 production phasein order to increase the rateof production. Themedium used forboth growth and H2 productionphases was only describedas a mineral salt mediumsupplemented with 3g/L yeastextract and 5g/L sucrose.Through direct contactwith theresearchers, it was laterdiscovered that the mediaused for growth wasLB mediasupplemented with glucoseand phosphate buffer. Inparticular, it wasmentioned thatglucose was used ratherthan sucrose because Citrobacterspecies are known tonot usesucrose as a carbon source.Due to the contradictorynature of these statementsand thelack of details in thepublished research paperson the composition of themedia, theresearch in this thesis investigatedthe effect of several differentmedia componentsongrowth and H2 productivityusing Ci amalonaticusY19.25Once a method for this two-phaseprocess was established,optimal conditionsforthe growth phase were investigated,including pH, temperature,and 02 and CO partialpressure. Optimal temperaturesand pH for growthwere found to be30-40°C and 5-8,respectively. The experimenton the effect of02 partial pressure was conductedin thepresence of 0.O5atmCO in the headspaceand varying02 partial pressures. H2productivity was shownto be affected by theP02 in the gas phaseand inhibited byP02above 0.4 atm. Consideringboth cell growthand H2 production,the optimal P02wasestimated to be 0.2-0.4atm(Jung et al. 2002).Another study morespecifically onthe 02sensitivity of hydrogenproduction activityof Ci amalonaticus Y19found that in wholecells, the deactivation ofhydrogenase by02 was reversible. On the contrary,COdehydrogenase activitywas not recoveredonce deactivatedby 02, and the onlyway torecover the activitywas to synthesize newCO dehydrogenase(Kim et al. 2003).The effect of CO-partialpressure(P0)in the growth phase on cellgrowth rateand H2 production activitywas also studied inthe range of 0.0to 0.5atmPco.Themaximum specificgrowth rate was shownto decrease linearlywith increasingPco. TheH2 productionactivity was foundto be almost negligiblewhenPc0 was zero but rapidlyincreased whenPco was raised to 0.05 atm. At higherPro,the activity decreaseduntilPco reached 0.3 atm at which timeactivity levelled off.The researchersattribute thelinear decreaseof the maximum specificgrowth rate withincreasingPo to aninterference withthe electron transportchain (Jung et al.2002). They alsoconclude thatthe presence ofCO during the growthphase was necessaryfor the inductionof H2production activity,and that the activitymight be developedduring the cell-growthperiod via a CO-dependantmechanism. They furthersuggest that the relevantenzymesfor CO-dependentH2 production aresynthesized duringaerobic growth andactivatedduring the subsequentanaerobic incubation.It seems unlikelythat this conclusionisaccurate because bacteriawill not expend energyproducing unnecessaryenzymes whenit is under desirablegrowth conditions.Furthermore, if theserum bottles aresealed inorder to contain COin the headspace,it is likely that thebatch cultivations willrun out ofair before the bacteriareach their fullgrowth potential.The effect of the timeat which the culturewas shifted fromthe aerobic growthstage to the anaerobicH2 production stagewas also investigated.The best resultwas26obtained when the culture was transferred after 12 hours of growth, which correspondstothe start of the stationary growth phase. When the shift was performedat longer andshorter times, the hydrogen production activity obtained in the anaerobic hydrogenproduction stage was significantly lower (Jung et al. 2002).The effect of temperature and pH were also examined duringthe hydrogenproduction stage. Optimal conditions were found to be between 30-40°C for temperatureand between 5.5-7.5 for pH. Above pH 9 or below 5.5, the hydrogenproduction activitydecreased rapidly and the stability of the system became very low.As previously noted, sucrose andlor glucose was used to supplementthe mediafor all of these experiments, thus adding another carbon source.Consequently, if therewas any remaining carbon source after the growth phase, any hydrogen productivityresult may not come solely from the effect of CO as a substrate. In orderto differentiatethe effect of the additional carbon source on the growth andH2 productivity of Ciamalonaticus Yl 9 from the effect of CO, experiments were performedin this researchproject using only CO as the carbon source added to LB media, aswell as using CO withadditional glucose in LB media.Another observation made by the researchers was on the effectof deletingtryptone from the media. Tryptone is hydrolyzed milk proteinand is thus made up ofmany essential amino acids that contribute to the complexity ofthe media. Jung et al.(1999) reported that while both cases exhibit high rates of CO consumptionand H2production, the media with tryptone-deletion showed muchhigher rates than the mediacontaining tryptone. From this result, they further concluded that the presenceof a carbonsource during the H2 production stage inhibits the utilization of CO inCi amalonaticusY19. Biologically, this result is troubling because tryptone consistsof an assortment ofamino acids, but cannot actually be considered a carbon source. The yeastextract in LBmedia would be more accurately the carbon source amongother things, and thus theeffect of the tryptone deletion observed must be attributedto another factor. The researchin this thesis addresses the confusion of this result by re-investigatingthe effect ofdeleting tryptone from LB media.The batch shake flask reactors have also been usedfor kinetic studies in order todetermine the kinetic parameters of Ci amalonaticusY19 (Jung et al. 2002). The data27follow a second order polynomialcurve rather than a straightline, indicating that thecellular system undergoes substrate inhibitionduring the water-gas shiftreaction. Thisobservation suggests that the Andrews modelwith the substrate inhibitionterm is moreaccurate than the Monod model for hydrogenproductivity of Ci amalonaticusY19 (Junget al. 2002). Andrews model contains non-competitivesubstrate inhibition:q=qmaxo2(14)K+F0+—-where q is the specific H2 productionrate (mmol/(g cell x h)),q,is the maximum H2production rate,Pco is the partial pressure of CO, K’ is the Monod constantwhen thesubstrate is gas (atm), and K1’ isthe substrate inhibition constant(atm). Jung et al. fittheir experimental data to Andrewsmodel and obtaineda of 266.18 mmol/(g cell xh), a K of 3.49 atm, and aK1 of 0.18 atm.Overall, some of the results presentedin this section serve as astarting point forthe research performed in this study,while others bring up interestingquestions, whichare further verified or challenged. These questionsinclude: is CO necessaryin the growthphase for the production of the keyenzymes used during H2production? Can CO be usedas the sole carbon source for H2production or is it necessaryto add sucrose andlorglucose? Does the deletion of tryptoneindeed increase H2 productivity,and if so, howcan this be more accurately explained?283.0 Scope and objectivesThe world’s oil usage perannum is projected to increaseby over 40% over thenext 25 years to meet our growingneed for energy, materials,and chemicals. In the nearterm, Canada can and most likelywill address this increaseddemand through moreaggressive harvesting of its oilsands and offshore reserves.However, these reserves arefinite and non-renewable,a fact that is driving an unprecedentedrise in retail pricesforfuels, growing interest in nationalenergy security, anddeep concerns aboutthe diversity,health, and sustainabilityof our global ecosystems.Our national energy policymusttherefore foster developmentof a broad range ofoptions to reduce vulnerabilitiestosupply disruptions whileprotecting the environment.We must find alternativeenergy andchemicals feedstocks andefficient processes tomaintain sustainable economicgrowthand reduce our dependenceon fossil fuels. One viableoption is to derivefuels, materials,and chemicals from lignocellulose,a chemically complexand renewable feedstockthatcan be grown on low-valuenon-agricultural landand therefore offers the advantageofnot competing with food production.This research focuses on biologicalhydrogen production usingthe water gas shiftpathway as catalyzedby Citrobacter amalonaticusY19, a chemoheterotrophicmesophilicbacterium classifiedas gram negative andfacultative. Relatively littleis known about theH2 production capabilitiesof Ci amalonaticus Y19,though its ability toproduce hydrogenanaerobically usingcarbon monoxide as the substratehas been demonstratedin the onepublished study todate (Jung et al. 2002).As the intent of this researchis to use thissystem as a modelfor evaluating the potentialand economics ofbiological hydrogenproduction, the followingobjectives are defined:1 • To find and evaluatea media that allows Citrobacteramalonaticus Y19to growaerobically.2. To utilize a gas representativeof low-grade syngas ina two-stage process toproduce H2 using the biologicalwater gas shift reaction.293. To determine therneffects of mediacompositionand growth conditionson thehydrogen productivityof the system, andrelate the H2productivity resultstoenzyme activity. Themedia modificationsinclude the presenceof CO duringthegrowth phase, theaddition of glucose,the deletionof tryptone, and theaddition oftrace metals to the mediaduring the hydrogenproduction phase.4. To define a strategyfor minimizing theinhibition of enzymefunction.5. To determine whetherthe mass transferof CO from thegaseous to the liquidphase is a limiting factoron hydrogen productivity.In order to accomplishthese objectives,extensive experimentalwork wasconducted includinga design of experiment(DOE) factorialdesign on ironand nickelconcentrations forobjective 3. The valueof the thesis is thustO add insight andelaborateon the limitedliterature availableregarding the watergas shift pathwayusing Ciamalonaticus Y19with an emphasison mass transfer andmedia alterations,as well as toassess the large scalefeasibility of thistechnology.304.0 Materials and methods4.1 Materials andmedia composition4.1.1 MicroorganismThe microorganism usedthroughout this studywas a pure strain of Citrobacteramalonaticus Y19, a chemoheterotrophicfacultative bacteriumisolated from an anaerobicwastewater sludge digesterin Korea. Citrobacter isa genus of gram-negativecoliformbacteria in the Enterobacteriaceaefamily. Ci amalonaticusY19 has been characterizedby 1 6S rDNA analysis,Basic Local AlignmentSearch Tool (BLAST),and anApplication ProgrammingInterface kit (API20E kit), which isa standardized micro-method for the identificationof unlcnown gram-negativebacteria. The BLASTsearchshowed that the 16S rDNAsequences of thebacteria previously knownas CitrobacterY19 were 99% identicalto those of both Cifarmeriand Ci amalonaticusin the GenBankdatabase. The APIbiochemical testkit showed thatthe general biochemicalcharacteristics ofY 19 were exactly the sameas Ci amalonaticus,and not Ci farmeri (Ohet a!. 2008). The bacteriumwas shipped from Koreaat room temperature inagar.4.1.2 MediacompositionSeveral media compositionswere investigated throughoutthe project, thusa basecondition is definedand the modificationsto this base conditionare described. It shouldbe noted that because theprocess has two stages,the media sometimesdiffers in the H2production stage fromin the growth stageif the culture was re-suspendedin between.The base media usedwas Lysogeny Broth (LB;also known as LuriaBertani)media (Table 3) supplementedwith 0.5 mL/L of PTM1trace salts (Table 4).The PTM1trace salts were pre-madeat the Michael SmithLaboratories (MSL).The trace salts werefilter sterilized,stored at room temperature,and covered with foilto avoid lightpenetration. TheLB media, along withall of the glasswareand the septaused to seal thebottles, was autoclavedat 121°C for 15 minutesin a Steris-AmscoCentury SV-136HPrevac Steam Sterilizer,and stored at 4°C.31Table 3— Lennox’s Luria Bertani mediacomposition (LB)CompoundDescriptionAmountSourceNameper L H20Pancreatic digest of1 Og BDBacto-TryptonecaseinBacto-Yeast Extractof autolysed5g BDExtract yeastcellsSodium Chloride5g 99.9%, Fisher(NaC1)Table 4 - PTM1 trace salts composition,pre-made from MSLCompound AmountCompound NameFormula per LH20Cupric sulfate, pentahydrateCuSO4-5H20 6.0gSodiumiodideNal 0.08gManganese sulfate, monohydrateMnSO4-H20 3.0gSodium molybdate, dihydrateNaMoO-2H0.2 gBoric AcidH3B00.02 gCobalt chloride, hexahydrateCoC12-6H00.5 gZinc chlorideZnC1220 gFerrous sulfate, heptahycirateFeSO4-7H2065 gBiotin (aka vitamin H orB7) C10H16N3S0.2 gSulfuric acidH2S045.0 mLModifications madeto the base media included thedeletion of tryptone fromthebase media, the addition of glucose(anhydrous, EMD chemicals),and the addition ofbuffer. In more detail, the deletionof tryptone was done duringthe H2 production stageand resulted in a media containing5g/L of yeast extract, 5g/Lof NaC1, and 0.5mLILofPTM1 salts. When glucosewas added to the base media,it was added at a concentrationof 5g/L. The buffer usedto stabilize the pH wasa 20mM potassium phosphatebuffer atpH 6.5. A 1M-potassium phosphatebuffer was made from200mL of lM-monobasicpotassium phosphate (99.5%,Fisher) and l4OmL of1M-dibasic potassiumphosphate32(99.5%, Fisher). Inthe instances whenthe reaction was buffered,0 .8mL of the I Mpotassium phosphatebuffer was added to the 4OmLLB media resultingin a 20mM bufferconcentration.4.1.3 Designof experiments (DOE)In a DOE, a factorial designwas completed to observethe effect of addingtracemetals to the base media.The trace metals investigatedwere iron (FeSO47H20,101.0%,Fisher) and nickel(NiCI26H0, 98.88%,Fisher) at three differentconcentrationsforeach. A full factorialwas effectuated onthis 2-factor, 3-levelexperimental design.Thebase amount of iron containedin the PTM1 saltswas taken into accountand the totalconcentrations are thenumbers reported inTable 5. A 1 Og/L nickelsolution and a 20g/Liron solution wereprepared as stock solutionsfor ease of additionto the fermentationbroth. The nickel and ironsolutions were filtersterilized beforebeing added to the mediafor the H2 productionstage.Table 5 - Metal ion concentrationsLevel Fe concentrationNi concentrationDesignation(mglL) (mgIL)0 32.501 12562.52 2501254.2 MethodologyThe experiments wereperformed as twostage batch reactionsin 1 65mL Wheatonserum bottlesmade of clear borosilicateglass. A 12 hour aerobicgrowth stage wasusedto increase the celldensity above 2gIL, at which time the culturewas switched toananaerobic environmentby purging the crimp-sealedbottles with a gasmixture of 40%CO in helium.334.2.1 Citrobacter amalonaticus Y19growthCultivations were performed at 30°C in an incubatorshaking at a speed of250rpm. The liquid working volume was 4OmL inserum bottles of 165mL total volume.0.5 mL of Ci amalonaticus Y19 glycerol stock (AppendixA) was used as inoculum in4OmL of LB media (unless modifications are specifiedotherwise). While growth wasalways done aerobically, the serum bottles wereeither sealed or open to air duringgrowth. For the experiments where the bottles weresealed, a rubber crimp seal was put inplace after inoculation, followed by an addition of 6.5mLof CO gas using a sterilized gastight syringe. It should be noted that for the growthcurve analyses of Ci amalonaticusY19 under sealed conditions, lOmL of CO wereinjected rather than the 6.5mL of COused for the experiments that were carriedon to the next stage. The bottles grownopen toair were covered with foil to avoid contamination.After the 12-hour growth stage, a lmL samplewas taken from the fermentationbroth to measure cell density. When pH was measuredafter the growth stage, a 5mLsample was taken from the liquid phase.Organic acid concentrations were only measuredat the end of the growth stage whenthe process was terminated at this point;therefore,the entire fermentation broth was furtherfiltered as explained in section 4.3.3.4.2.2 Hydrogen producfionThe anaerobic H2 production stagewas started immediately after the 12-hourgrowth stage. Once the cell density was measured, dependingon the specific experiment,any additional media components suchas nickel, iron and buffer were addedto thefermentation broth. For example, in thenickel and iron factorial design experiments,0.5mL of the filtered lOgIL nickel solutionwas added with a pipette directly to theliquidbroth at this stage for the highest nickel concentrationof 1 25mg/L. Bottles that were opento air during growth were thencrimp sealed with sterile septa once all the additionalmedia components were added. The headspaceof each reactor was then purgedwith a40% CO in He gas mixture (v/v)for two minutes in order to remove allof the air andensure that no oxygen was present.The purging was done continuously throughtheseptum with one needle connecteddirectly to a gas cylinder containing pre-mixed40%34CO in He (vlv), and another needleof the same gauge size whichis not connected toanything allowing for thegas to circulate. A pressureof 6 psig is sustained inthe linefrom the cylinder with a regulatorto maintain a constant gasflowrate. The time at whichthe bottles are purged representstime zero for all hydrogenproduction graphs. Thepurged serum bottles were placedin a shaking incubatorat 30°C and 250rpm forapproximately 100 hoursor until all the carbon monoxidewas converted intohydrogenand carbon dioxide. Gas phasemeasurements of H2,CO, and CO2 concentrations weretaken every 10-12 hours dependingon the pace of CO conversion.For the experiments where pressurewas increased above 1 atmosphere,a pressuregauge (Omega engineering,0-1 O0psi, Connecticut)inserted through theseptum with aneedle was used to monitorthe pressure increase in thebottle. After the 2 minutesofpurging, the unconnected needlewas removed allowing the pressureto build by pumpingmore of the 40% CO in Hegas mixture (v/v) into thebottle.4.2.3 Re-suspension procedureIn specified cases, cellswere re-suspended in freshmedia between the growthandhydrogen production stages.After the 12-hour growthperiod, the cell cultureswere spundown at 5500rpm for10 minutes usinga Fisher scientific 5810Rcentrifuge. Thesupernatant was discardedand 4OmL of fresh mediawas added to the cells.Cell densitywas always measured before andafter re-suspension.4.3 Analytical andmeasurement techniques4.3.1 Cell density andpHSamples were collectedfrom the fermentation mediumfor monitoring theconcentration of Ci amalonaticusYl 9, and pH. 1 mLsamples were collectedfor celldensity measurements and5mL samples forpH. Two UV/visible spectrophotometerswere used throughout thestudy: a Milton Roy Spectronic20D+, anda Pharmacia BiotechUltrospec 1000. The cellgrowth was measuredin terms of the optical density(OD) at600nm. The lmL samplescollected beforeand after the H2 productionstage of theexperiment were dilutedin order to keep the ODmeasurement belowan absorbance of350.9. In order to find the conversionfrom optical density to drycell weight, Ciamalonaticus Y]9 was grown ina serum bottle open to air in 4OmLLB mediasupplemented with 0.5mL/L PTM1 salts. Duplicatebottles were incubated at 30°Cand250 rpm for 14 hours. The OD wasfirst measured, and then the broth was centrifugedat14,000rpm for 5 minutes. The OD of thedecanted supematant was measuredin order toensure no cells remained in the liquid.The cells were dried in an ovenat 70°C for 12hours before measuring the dry cell weight.The dry cell weight conversion wasfound tobe 0.48 ± 0.01 g DCW IL /unit of OD.The pH meter used was a VWR SB2O sympHony.pH was measured after thecompletion of the H2 production phase,and occasionally in between thegrowth and112production stages.4.3.2 Gas concentrationsGas phase analysis involvedmonitoring the concentrations ofH2, CO, and CO2formed during the fermentationprocess using a gas chromatograph(GC). The GC used isa Varian model 3800 equipped witha thermal conductivity detection (TCD)system and a6’ CTR I packed column by Alltech(Calgary, Alberta). The CTR Ipacking is made up ofconcentric columns, the outer columnis packed with an activated molecularsieve and theinner column is packed witha porous polymer mixture. The operationaltemperatures ofthe rear valve, injector, and detectorwere all 120°C. For112 detection, nitrogen was usedas the carrier gas with a flow pressureof 5psi and a column oven temperatureof 35°Cheld for 4.5 minutes. The retentiontime of H2 under these conditionswas around 2.9minutes. For CO and CO2 detection,helium was used as the carriergas with a flowpressure of 5psi and acolumn oven temperature of 90°Cheld for 12 minutes. Theretention times of CO and CO2were approximately 9.0 and 1.5minutes, respectively.Samples of 0.5 mL in volume were takenfrom the headspace of thecrimp-sealedserum bottles with a gas tightsyringe and manually injected intothe CTR I column. Thesoftware used to communicatewith the GC was Varian’sStar chromatographyworkstation System Control version6.41 and to acquire areas underthe curves wasVarian’s Star chromatography workstationInteractive Graphics version6.41.36Calibrations were performed forH2, CO, and CO2 using 2% H2in N2 (v/v), 40% CoinHe (v/v), and 2% CO2 in He (v/v)as standards, respectively (AppendixA).Injecting the same sample severaltimes tested the repeatabilityof the GCmeasurements. The results showeda very high repeatability withan average standarddeviation of 2% of the total value.4.3.3 Organic acid concentrationsA Waters High-PerformanceLiquid Chromatography (HPLC)system equippedwith a 2487 Dual absorbancedetector and an IC-Pak ion exclusioncolumn was used todetermine the presence andconcentrations of small acidssuch as lactate, acetate,andformate. Samples were preparedby first centrifuging the fermentationbroth at 5500rpmfor 10 minutes, then filteringthe supematant witha 0.22jim syringe drivenfilter beforemanual injections of 5OjiL intothe HPLC. The eluentused was 13mM H2SO4(95-98%,Fisher). The software usedto communicate withthe HPLC pump and detector,and toacquire areas under thecurve was Waters’ Breeze version3.20.Organic acids were presentat several retention times:8.3, 9.0, 9.8, 11.8, 13.4,and14.5 minutes. The elution timesof 8.3, 9.0, 9.8, and 11.8minutes correspondedto lacticacid, formic acid, acetic acid,and fumaric acid, respectively.Even though a widerangeof compounds were tested,the compounds with 13.4 and14.5-minute retentiontimeswere not identified. Itis assumed that these compoundshave a higher electricalaffinityfor the IC-Pak ion exclusioncolumn. Calibrations wereperformed for some ofthe knownacids using pure chemicalsunder their sodium salt form:sodium acetate(99.0%,enzyme grade,Fisher), and DL-lacticacid sodium salt (98%, Sigma)(Appendix A).375.0 Results and discussion5.1 Two-stage processfor H2productionfrom CO5.1.1 Growth stageIn order to verify the necessity of CO during the growth stage, two conditionswere investigated: a completely aerobic system where the serum bottle wascovered onlywith aluminum foil, and a crimp-sealed aerobic system that contained 8% CO(v/v) in thegas phase, with the remainder being air (Figure 5). All experiments were performedin ashaking incubator where the temperature and agitation rate were maintained constantat30°C and 250rpm, respectively.4 —______________________________________________3.522.504-4-20001.50010.500 5 10 15 2025Time (hours)Figure 5 - Citrobacter amalonaticus Y19 growth curve under twoconditions. Symbols: -i-, cellconcentration under completely aerobic conditions; -a-, cell concentration under sealedaerobicconditions with 8% CO (v/v) present in the headspace. Symbolsrepresent the experimental data andlines represent the best-fit model obtained using CurveExperts 1.3.38After the lag phase and during the exponentialgrowth phase, from 2.5 to 4.5hours, the specific growth rates for the openand sealed conditions were 0.84 and 0.74 W’,respectively (see Appendix B for logarithmic growthcurve). Following the exponentialgrowth phase, there is a linear growth phasefor the time period from 4.5 to 7 hours. Thegrowth rates during this linear growth phase are0.54 h’ for the open condition, and 0.45h’ for the sealed condition. At 7 hours, the deceleratedgrowth stage began, followedbythe stationary phase at around 12 hours. The maximumcell densities obtained for theopen and sealed conditions were 3.1 and 1.9 gIL, respectively.These results indicate thatwhile the growth patterns were almost identical priorto the 4-hour mark, the completelyaerobic conditions allowed Ci amalonaticus Y]9to grow to higher cell densities. Thissignifies that while the CO concentration in thesealed serum bottles is not inhibitorytocell growth, there is likely a lack of oxygenafter 4 hours. When sealed, the bacteria runout of air, which prevents cell growth to itsfull capacity.The concentration of organic acids wasalso measured before and after growth forboth the open and sealed conditions. The effect ofbuffering the growth at a pH of 6.5with 20mM potassium phosphate was also considered(Figure 6). While other organicacids were present due to the complex natureof the LB media, lactate and acetate werethe only components that increased significantlyin concentration during the growthstage. Lactate was produced during thegrowth stage to a final concentrationof 4 g/Lafter 12 hours of growth whether thesystem was open or sealed. Under openconditions,the concentration of acetate was 2.3g/L, whilesealed conditions resulted in acetateconcentrations up to 3 .4g/L. This increasein acetate concentration between the openandsealed conditions, in the absence of buffer,was statistically significant at a 95%confidence level. This suggests that acetate may beproduced more rapidly under sealedgrowth conditions once the culture ran out ofair and became anaerobic. When the systemwas buffered, the concentrations of lacticand acetic acid decreased proportionallyfor allconditions.In the previous studies on this organism (section2.4.3), Citrobacter amalonaticusY19 grew with a specific growth rate of0.7 h’ to a cell density of 2gIL in thepresence ofa CO-air (20:80, v/v) mixture, while acompletely aerobic condition was notreported(Jung et al. 1999b). This culture wassealed and thus may have run outof oxygen after a39certain length of time, which wouldexplain the lower cell densityof 2g/L as comparedtothe cell density of 3.1 g/L obtainedunder completely aerobiccondition (Figure 5).The effect of CO contentin the gas phase duringthe growth stagewas alsopreviously studied, andthe researchers (Jung et a!. 2002)determined that thehydrogenproduction activity was almostnegligible when no COwas present in the growthstage.The conclusion made basedon this result was that thepresence of CO during thegrowthstage induced the synthesisof the relevant enzymesthat were later activatedin the H2production stage (Junget al. 2002). As explainedin section 2.4.3, this resultwastroubling because bacteriahave a tendency to onlyproduce enzymes that areimmediatelynecessary for their survival.Since the bacteria wereunder desirable growthconditions,the production of CO dehydrogenaseand hydrogenase that wereto be activated in a laterstage seemed unlikely. Neitherthe previous study, northis study created an assayforthese two enzymesto confirm or deny theirpresence during the growthstage. One suchFigure 6 - Concentration of lactateand acetate, as well as pH afterthe growth phase. Hashed boxes,lactate; dotted boxes, acetate.Error bars represent the averageof 2 measurements.40assay would be necessary toassess the stage at whichthe CO dehydrogenaseandhydrogenase enzymes are produced.In this study, it has beenshown that the sealedgrowth condition with8% CO (v/v) present in air decreasedthe growth rate, andconsequently, the final cell densityattained. This resultwas most probably attributedtothe fact that the serum bottleswere sealed, and notto the presence of CO duringgrowth;however, this was not verifiedwith dissolved oxygen (DO)measurements. It shouldbenoted that these results,along with the increasedsimplicity of the reactorconditionswhen open to the atmosphere,make the completely aerobiccondition favorableover thesealed condition. The effectof these growth conditionson the H2 productivityin theanaerobic phase will be discussedin section 5.3.5.1.2 Hydrogenproduction stageBecause Ci amalonaticusY19 does not produce hydrogenaerobically, a two-stageprocess was suggestedwhere the bacteria are firstgrown aerobically followedby ananaerobic hydrogen productionstage. After 12 hours of growth,the serum bottles werecrimp-sealed and purgedfor two minutes with a gasmixture of 40% CO in helium(v/v).The hydrogen production phasewas carried out until theculture used all of theavailableCO substrate. Figure 7shows the evolutionof CO, H2 and CO2 forthe H2 productionstage of a buffered systemwhere growth was opento air and the cells werere-suspendedin fresh media after thegrowth stage. Note thatH2 concentration curvesfor otherconditions (all experimental conditionsinvestigated) are shownin Appendix B.The increase in H2 andCO2 concentration wasproportional to the decreasein COconcentration as expectedfrom the water-gas shiftreaction:CO + H2O — H2 + CO2AG° = -20 kJ mol’ (1)The zG° is reported at ambienttemperature and pressure.The conversionof CO into H2and CO2 was stoichiometricallyconsistent with thetheory. Also, thethermodynamics ofthe reaction suggest thatall of the CO willbe converted since the reactionis shifted to theright, and indeed, theCO is depletedbeyond the detectionlimit of the gaschromatograph. The molaryield of H2 on CO was70 ± 4% for most conditionsthat didnot exhibit any inhibitioncharacteristics. This suggeststhat some of the CO substrateisused to produce by-products,possibly in the form oforganic acids.4160— 50• 455040• 30________25——COconcentration6—4.— Hydrogen concentration20o—i-- C02 concentration15____________________________1010:40 6080 100Time (hours)Figure 7— CO, CO2 and H2 concentrationsduring the H2 production phasefor a buffered systemwhere growth was open to air. Purgedwith 40% CO in He (v/v) at time0. Symbols: -+-, H2; -•,CO; - A , CO2. Data points representthe average of 2 runs andthe error bars represent thestandard deviation.In order to convert H2 concentrationsinto H2 productivities, theslope betweeneach set of consecutive datapoints was calculated.This slope represents theH2productivity at the average time betweenthese two data points (Figure8). It is known thatwhile batch production is veryhelpful for initial system characterization,,it is inefficient.Continuous H2 productionwould thus be necessary to ultimatelycommercialize this H2producing system. Therefore,only the maximum H2productivities attained arerelevantsince the decreasein productivity was simply due tothe gaseous substrate, CO,beingdepleted. The time at which thesemaximum productivities were achieved(tm) is alsoshown because it indicates theamount of time necessaryto produce the enzymes used forH2 production. It shouldbe noted that if the lifetime of theenzyme activity is long,attaining a shortertmax might not be an important parameter. The maximumproductivityis illustrated in the formof bar graphs for the remainderof this discussion. While theslope of consecutive datapoints gave the time evolutionof H2 productivity, the maximumI-J0EC030I00 2042.x-J0EE000L..4-’E0>productivities were more accurately determinedmathematically by using CurveExperts1.3 to find the best-fit model of H2 concentrationversus time, when sufficientdata wasavailable. The best-fit model for the lag andH2 production phases used wasa degreepolynomial:[H2]= a + bt + Ct2 + dt3(15)Setting the second derivative of this equationequal to zero, and solving forthe1stderivative of H2 concentration provided themaximum productivity.1.81.61.41.21.00.80.60.40.20.0-0.20 20 4060 80 100Time (hours)Figure 8 — Volumetric H2 productivityfor a buffered system where growthwas open to air. Purgedwith 40% CO in He (v/v) at time 0.Data points represent the average of2 runs and the error barsrepresent the standard deviation.Organic acid concentrations weremeasured after the H2 productionstage for botha buffered and non-buffered system.Unlike the growth stage, therewas no increase inlactic acid production duringthe anaerobic H2 productionstage. However, acetic acidconcentration increased from 0g/L in LB media to 3 gIL inthe buffered and non-buffered systems alike. Formate was alsoproduced in the H2production stage wherethere was none in pure LB media,and an unknown organicacid that eluted at 14.5minutes from the HPLC also increaseddramatically in concentrationduring this stage.43Although the composition of this organic acid that elutesat 14.5 minutes is unknown, it isassumed that it has a high electrical affinity to theIC-pak charged solid separationmedium in the column, as it is the compound that isretained the longest.The maximum H2 productivities for all the conditions throughoutthis researchproject ranged from 0 to 1.5 mmol H2! (L x h) (or 0 to0.7 mmol H2/(g cell x h)) as willbe detailed in the following sections. While this suggests thatmany of the conditionsinvestigated had a significant impact on the H2 productivity,it is also a lowerproductivity than what has been previously reportedfor this organism. The researchgroup that has investigated using Ci amalonaticusY]9 for the water gas shift reactionreported a maximum specific H2 production activity of 27.1 mmolH2! (g cell x h) (Junget al. 2002). Using the cell concentration range of 0.1to 0.4mgImL reported for themeasurements of specific H2 production activity,the maximum volumetric H2productivity they attained was between 2.71 and10.84 mmol H2! (L x h). Thediscrepancy between these results of up to one orderof magnitude can be explained byseveral reasons including the different media useddue to a lack of details in thepublications, and an alternate method of measuring theH2 productivity.The exact composition of the media wasnot specified in the literaturepublications on this organism, and the papers thatwere referred to regarding the mediaresulted in a fruitless effort to find the exactcomposition used. Direct contact with theauthors of the publication resulted in vague descriptionsof media compositions thatsometimes conflicted with what was reported in the publications.The conflict mainlyregarded the supplementation of the mediawith either glucose or sucrose. Sucrose wasreported in the publication, but an email communication suggestedthat Ci amalonaticusY19 did not utilize sucrose but rather glucose. The deletionof tryptone from the mediawas also declared in an earlier publicationbut not mentioned in a later paper (Jung et al.2002, Jung et al. 1 999b). These manyunknowns regarding the media composition usedfor the aerobic and anaerobic stages of the reactioncould be one explanation for thediscrepancy in H2 productivities.The differing methods of measuring H2 productivity seemedto be the major causeof the discrepancy between the results reportedhere and the results from the Koreanresearch group. According to the previous researchpapers, specific H2 production44activity was determined as a point measurement aftera 24-hour induction phase in 20%CO. The cells were harvested after 24 hours and re-suspended ina buffer solution at aconcentration between 0.1 and 0.4 mg/mL. A lmLsample of this cell suspension ischarged with a CO-Argon gas mixture in the headspaceand the H2 concentration is thenmeasured for an hour. These point measurements at 24 hours arenot as insightful into thekinetics of the reaction as the continuous measurements donein this research. Re-suspending the cells at a different concentration and with freshCO may also affect themeasurement accuracy.Overall, this discussion concludes thata comparison between the volumetric H2productivities obtained in this research withthe specific 112 production activities obtainedin the previous publications is invalid. Ratherthan comparing values with previouspublications, the H2 productivities obtained are evaluatedin comparison to a base casepresented here, and each experimental condition wasrun in parallel with standards.5.2 Effect ofCO concentration during H2productionphaseIn order to select the best concentration of COto use in the experimental runs,two factors were considered: the possibility of substrateinhibition at high levels of CO,and the typical amount of CO in low-grade synthesisgas. In order to assess whethersubstrate inhibition takes place, experiments wereperformed at several initial COconcentrations ranging from 7 to 100% CO (v/v), withthe remainder of the gas phasebeing helium. The cultures were grown open to the atmospherein LB media, and thensealed and purged to ensure the oxygen was completelyremoved, without re-suspensionbetween the growth and H2 productionstages. Helium was used as the inert gas in thesystem because it had the most distinct conductivity,and thus retention time, in the gaschromatograph when considering all the gases measured(CO, CO2 and H2). For the rangefrom 7-60% CO, the serum bottles were first purgedwith pure helium, and the headspaceratio was then adjusted by removing the appropriateamount of helium and adding COusing a gas-tight syringe to ensure the bottlewas not pressurized in this process. Figure 9illustrates the results obtained for this range of COconcentrations.This figure illustrates that while the H2 productivityafter the lag phase ofapproximately 15 hours seemed relatively constantthroughout this range of45concentrations, as seen fromthe slope of the curves, the H2production stage lastedlongerfor higher CO concentrations.This is simply because at lowinitial CO concentrations,the substrate was exhausted faster.Figure 9 also shows noevidence of substrateinhibition up to initial concentrationsof 60% CO.Figure 9 - Batch evolutionof H2 concentration for varying initialCO concentrations. COconcentrations vary from 7to 60% CO in Helium (vlv). Symbols:-i-, 7% CO; -R-, 14%CO; - A -,27% CO; -•-, 40% CO;-h-,60% CO. Symbols represent the experimentaldata and lines representthe3fhdegree polynomial best-fit curves.An estimate of maximumH2 productivity was obtainedby fitting the H2concentration versustime data to a 3’ degree polynomialequation and setting the2ndderivative of this fitted equationto zero as described insection 5.1.2 (Figure10). Sincethis screening experimentwas used to determine whichconcentration of COshould beused for the remainder ofthe tests, only one serum bottlewas run for each condition.First, it should be noted thatsince the maximal H2productivity at the 6.8% COconditionwas reached so quickly, only6 data points were used to fitthe 3’ degree polynomial,andonly three of these data pointswere collected during theH2 production phase, decreasingthe reliability of this modeland the validity of the maximalH2 productivity. Lookingatthe remaining three conditionsin Figure 10, tripling the concentrationfrom 13.6% CO to403510EE20015100(‘15I0-50 2040 60 80100Time (hours)4640% CO (v/v) resulted in nearly doubling the timeat which the maximum productivityoccurred from 20 to 38 hours, but the maximum H2 productivityonly increased by 0.1mmol/ L liq x h, or 25%. The total time it took to convert COincluding the lag phaseranged from 26 hours for 7% CO to 66 hours for 40% CO. Thisshows that using higherinitial CO concentrations allows for significantly longer timeperiods to monitor theconversion of CO to H2,which is more informative because thekinetics can be observedfor longer..xg0•5-I0E0.4E> 0.320.C,’I0.1Figure 10 - Maximum H2 productivity and the timeat which the maximum productivities arereached for initial CO concentrations ranging from 7to 40%CO in He (v/v).For concentrations equal to or above 60%, the reversemethod was performed toattain the desired concentrations. Serum bottles werefirst purged with 100% CO for twominutes, and the headspace ratio was then adjustedby removing CO and adding purehelium (Figure 11). This experiment showed decreasedH2 production rates for bottlesthat were first purged with 100% CO, suggesting thatthis purging process inhibits theH2-producing enzymes. The F{2was produced at a much slower rate and theCO was notcompletely exhausted after 105 hours. Perhapspurging the culture with pure CO drives0.70.607% 14% 27%40%CO concentration in the gas phase(%)47the CO, which has a very low solubility in water, into the media, resulting in anunderestimation of the initial CO concentration. While this substrate inhibition at veryhigh levels of CO is present, it is not a concern for the biological H2 production process,as these high levels of CO are not in the targeted range of initial CO concentrations,which is under 40% CO (v/v).Figure 11 - Effect of the purging gas used (CO or He) on the H2 concentration evolution. Initial COconcentration adjusted after purge from 40-100% (v/v). Symbols: -4—, 40% CO, purged with He; -,60% CO, purged with He; -i-, 40% CO, purged with CO; -•-, 60% CO, purged with CO; -‘v-,80% CO, purged with CO; -b-, 100% CO, purged with CO. Symbols represent data points, andlines connect the symbols for ease of visualization.Low-grade synthesis gas is the desired substrate for this system as it is a lowchemical energy feedstock that can be considered a waste stream. As previously defined,low-grade syngas has a low CO concentration, under 20% CO, and is at a lowtemperature, close to ambient temperatures. While lower than 20% CO is the targetedrange of initial CO concentration, this screening experiment showed that increasing theinitial CO concentration to 40% significantly increases the time period available toobserve the conversion of CO to H2, while not reaching the limit of substrate inhibition.4035302520w 15C.,0C.10I500 20 40 60 80 100Time (hours)1204840% CO was thus considered optimal as it did not inhibit the reaction, but allowed forsufficient observation of the reaction kinetics. As for the temperature, the substrate wasreceived and dispensed into the system at ambient temperatures and heated up to 30°C inthe shaking incubator. While syngas with less than 20% CO can still be efficientlyprocessed by a conventional WGS using a metal catalyst, it would not be economicallybeneficial to heat up syngas that is at ambient temperatures. In conventional WGS, LTSreactions operate between 200 and 260°C, and HTS reactions operate between 300 and500°C. For situations where syngas is at ambient temperatures, the biological WGS canthus be very practical.The method of purging the serum bottles with pure gasses and adjusting theconcentration to the desired ratio afterwards caused several problems, including asignificant effect from the purging gas used, but also compromising of the septa used toseal the serum bottles by repeated injections with needles. While the data is not shown,several data sets had to be discarded because the many holes created in the septa byrepeated needle injections caused air to enter the bottles. This end to the anaerobicconditions in the serum bottle caused an abrupt halt to H2 production. All experimentshereafter were thus purged with a pre-mixed 40% CO in He mixture (vlv) directly from agas cylinder to minimize experimental problems such as holes in the septa, and tomaximize the observation time of the reaction kinetics without reaching a level ofsubstrate inhibition.It should be noted that syngas generally contains not only CO, but also manyother components such as CO2,H2, and minor amounts of other gases such as CH4,N2,and H2S. There is a large variation in syngas. composition depending on the source fromwhich it was produced. The gas used as substrate for the experiments in this researchcontained only CO and inert gas at the start of the anaerobic phase of H2 production. Itwould be interesting to investigate the impact of the other components of syngas on thebiological WGS reaction. Since H2 and CO2 are on the right hand sideof the WGSreaction, it is speculated that the presence of these gases might decrease the efficacy ofthe process by product inhibition. At ambient temperatures, the WGS is shifted to theright, so the magnitude of the product inhibition is unknown, but may only be quite small.495.3 Effect ofmedia compositionThe H2 productivities obtained thus far in the research were low, and so effort hasbeen put into investigating various means of increasing these productivities. One possiblemethod of increasing the H2 productivity was to develop a media optimized for H2production. The initial parameters were selected based on literature data using Ciamalonaticus Y19 (Jung et al. 2002 and 1999b). These parameters include the presence ofCO in the headspace during the growth stage, the addition of a carbon source such asglucose or sucrose to the media, and the deletion of tryptone from LB media. Asexplained in section 2.4.3, it is speculated that these media modifications do not have aspositive of an effect on H2 productivity as is claimed in the two literature studies. Themaximum H2 productivities obtained experimentally for these modifications areexpressed in Figure 12. The productivities were found using the maximum slope ofconsecutive data points rather than the3iddegree polynomial best-fit approach becausethe small amount of data points would produce inaccurate curve fitting with insufficientdegrees of freedom. The H2 concentration curves for these conditions are in Appendix B.Figure 12 - Effect of addition of CO during the growth phase, addition of glucose in the H2production phase, and deletion of tryptone in the H2 production stage on H2 productivity comparedto an average productivity found throughout the research experiments. Bars represent the average of2 runs and the error bars represent the standard deviation..rx0-J0EE4-z2C,’I1.41.21.00.80.60.40.20.0IControl CO duringgrowthCO during Glucosegrowth,wlbufferTryptone Tryptonedeletion deletion,wlbuffer50The maximum H2 productivity attained for a re-suspended system in LB media,where the growth stage was completely aerobic, was approximately 1.2 mmol H2!(L x h)with pH 6. This productivity serves as a control to compare with the other productivitiesobtained. The cell density at the beginning of the H2 production stage was between 2.0and 2.3 g cell!L liq for all of these conditions except for the conditions with CO presentduring the growth phase. As explained in section 5.1.1, the sealed growth condition withCO present resulted in a lower cell density. The cell density at the start of the H2production stage for both sealed conditions, with and without buffer, was approximately1.4 g cell/L liq.The sealed growth condition with CO present reduced H2 productivity in the H2production stage by a factor of 50 to 0.023 mmol H2! (L x h). The pH was measured afterthe H2 production stage when CO was present during sealed growth and was found to be5, which is below the optimal range of 5.5-7.5 (Jung et al. 2002). With the addition of20mM potassium phosphate buffer at pH 6.5, the maximum H2 productivity increased to0.18 mmol H2 /(L x h), still a 7-fold decrease from the average number. The pH for thisbuffered solution was 5.3, still below the optimal range, thus suggesting that a higherbuffer concentration should have been used. These results suggest that the sealed growthcondition with CO present produced some acidic metabolites at high concentrations thatdecreased the pH significantly. As shown in Figure 6, the concentration of lactic acid wassimilar for both the opened and sealed growth conditions; therefore, acetic acid, whichwas produced at higher concentration in the sealed condition, may be the cause of the pHdecrease.It should be noted that the experiments discussed above with CO present in thegrowth phase were not re-suspended in fresh LB media between the growth and H2production phases. One such experiment, with re-suspension, was completed to verifythat it is the acidic metabolites produced during sealed growth that decrease the H2productivity (Figure 13). When the media is re-suspended between phases, the sealedgrowth condition with CO present does not have a significant impact on H2 productivity.This confirmed that it is the metabolites produced during the growth stage that inhibit theproduction of H2 or decrease the pH to an inhibitory level.5150——LB growth, re-suspension in LB—S—Growth sealed with CC, no resuspension—*--Growth sealed with CC, buffered, no resuspension.—-—Growth sealed with CC, resuspension in LB. II0 20 40 60 80 100 120Time (hours)Figure 13- Effect of sealing the growth stage with 6.25mL CO in the headspace under severalconditions. Symbols: -+-, LB growth, re-suspended in LB; -.-, Sealed LB growth with CO; -A,Sealed LB growth with CO, buffered; -.-, Sealed LB growth with CO, re-suspended in LB. Symbolsrepresent average of 2 experimental runs and error bars represent the standard deviation. Linesconnect the symbols for ease of visualization.These results oppose the previous study’s finding, explained in section 2.4.3,where the presence of CO in the growth phase was necessary to achieve any H2production at all (Jung et al. 2002). The previous rationalization was that the COdehydrogenase and hydrogenase enzymes were synthesized during aerobic growth via aCO-dependent mechanism. Bacteria have a tendency to only produce what is necessaryfor their survival, and under desirable growth conditions, it seems unlikely that theseunessential enzymes are synthesized. The claim that CO is necessary during the growthstage for later H2 production is thus refuted, as not only is H2 productivity higher whenthe growth stage is completely aerobic, but higher cell densities can also be attainedunder this condition (Figure 5). These results show that the sealed growth condition withCO present not only increases the complexity of the growth stage, but also producesinhibitory metabolites that have a negative impact on 112 productivity. These results also4535E30C25200(3C0(3tb5052provide insight into the sensitivity of Ci amalonaticus Y19 to pH. pH significantlydecreased productivity when it dropped below 5.5, thus suggesting that the pH should becarefully controlled for this process.The impact of adding an external carbon source on H2 productivity was alsoinvestigated. Opposing statements between direct contact with the authors of thepublication and what is printed in the publication created confusion about which carbonsource was present, glucose or sucrose. While there is still confusion about the specificcarbon source used in the literature study, the conclusion remains that an external carbonsource was added to the growth media for all of the experiments performed in theprevious study.The results from these experiments show that when glucose was added to the LBmedia at a concentration of 5g/L, there was no H2 production whatsoever. It was alsoobserved that the CO was not utilized and remained at its initial concentration throughoutthe H2 production stage. Strong catabolite repression seemed to be taking place becauseglucose was the preferred substrate over CO. This suggests that the Ci amalonaticus Y19used the glucose for continued growth, even under anaerobic conditions, which wasverified with cell density measurements (Figure 14). Indeed, the cell density increasedwhile CO concentrations remained constant in the reactor throughout the 100-houranaerobic stage.The discrepancy with the previous study regarding addition of carbon sources tothe LB media may be a result of the time at which the glucose was added. Jung et al.(2002) suggest the carbon source is added to the media at the start of the growth stage, soit is possible that the sucrose (or glucose) is exhausted before the start of the H2production phase. In any case, this experiment has demonstrated that external carbonsources are unnecessary and inhibitory to H2 production. As carbon sources such asglucose and sucrose are expensive, this finding also decreases the cost of the media.5345• LB with re-suspension0 Glucose present in growth and H2production stages43.5(3Ca)01.5I0Time (hours)96Figure 14 - Effect of glucose in cell density measured before andafter the hydrogen productionphase. Bars represent the average of 2runs and the error bars represent the standard deviation.One literature study suggestedthat deleting tryptone before the anaerobic stageincreased H2 productivity (Jung et a!. 1 999b).These researchers said that it is thepresence of a carbon source during the H2 productionstage that inhibits the utilization ofCO in Ci amalonaticus Y19. As tryptone is nota carbon source, but rather a source ofamino acids because it consists of hydrolyzed casein,deleting it should not increase theH2 productivity on CO. In the experiments conducted,tryptone deletion resulted in no H2production (Figure 12). This result suggeststhat Ci amalonaticus Y]9 could beauxotrophic for certain amino acids, which areprovided by the addition of tryptone. Inorder to confirm this, the pH was checked andfound to be 5, which is below thesuggested optimal pH range (5.5-7.5). Buffer wasadded to see whether it was the effectof tryptone deletion or the pH that ceased theproduction of H2. Adding 20mM phosphatebuffer at pH 6.5 increased the pH to 6 andreversed the negative effect seen previously.While the buffered tryptone-deleted condition brought theH2 productivity back toaverage levels, it did not increase H2 productivity, thusopposing the previous literaturefinding. The HPLC results showed that while fresh tryptone deletedmedia had a similarT54composition to LB media, the concentration of all the organic acids after the H2production stage were lower with tryptone deleted LB media, as compared to LB media.These experiments suggest that tryptone acts as a buffer, and can be converted intoorganic acids during the anaerobic stage. However, tryptone is not necessary to H2production and Ci amalonaticus Y19 is not auxotrophic for components of tryptone.From the effects observed in this section regarding media components, onefinding stands out: pH. For the sealed growth conditions containing CO and the conditionwith tryptone deletion, pH dropped below 5.5, which caused a significant decrease inproductivity. pH thus had a strong effect on H2 productivity of Ci amalonaticus Y19, andshould be controlled carefully throughout the process. For this study, pH was partiallycontrolled with a 20mM phosphate buffer at pH 6.5, but a more precise method,particularly if a continuous is used, would be preferable.5.4 Effect oftrace metalsDesign of experiment was used to see whether the addition of several trace metalswould have an impact on hydrogen productivity. Nickel (Ni) and iron (Fe) were themetals investigated due to their predicted impact on the two enzymes responsible forproducing hydrogen in this water gas shift reaction: carbon monoxide dehydrogenase andCO-induced hydrogenase. A full factorial design experiment with 3 levels of each metalwas used to ensure an interaction effect between the metal ions was not overlooked in theanalysis. The levels were based on typical concentrations of Fe, and zinc rather thannickel, in PTM 1 salt solutions. Zinc was used because it has similar properties to nickeland nickel is not present in PTM1 salts. These levels were 32.5, 125 and 250 mg/L forFe, where level 0 is not zero because of the basal level of Fe in the LB mediasupplemented with PTM1 salts, and 0, 62.5 and 125 mg/L for Ni. Most conditions wererun in duplicate, but the four conditions that demonstrated an increased H2 productivityafter the initial data set were run in quadruplicate, making the total number of runs anddata sets 26, rather than the 18 attributed to running duplicates of the full factorial design.The maximum volumetric 112 productivities, and the time at which these productivitieswere obtained, are shown in Figure 15 for each condition in the factorial design.551.4Figure 15 - Maximum H2 productivity for all the combinations of levels in the factorial design on Feand Ni concentrations. Data bars represent the average of 2 or 4 runs depending on the condition,and the error bars represent the standard deviation. The average time at which the maximumproductivity is attained is shown above the data bars.The trend observed in the factorial design was an increase in H2 productivity withincreased Ni concentration, but only when the Fe was at level 0 (32.5 mg/L). At increasedFe concentrations, there was no obvious effect on productivity, possibly due to aninteraction effect between Ni and Fe. While Ni seemed to increase H2 productivity, Fe athigh concentrations might cause the productivity to decrease, thus making the effectunclear at mixed concentrations. Another observation is that while the time at which themaximum productivity is reached was most often in the range of 33 to 36 hours, severalconditions had lower times, suggesting that the active form of the enzyme was producedmore rapidly. These conditions included all the conditions at the highest Ni concentrationof 125 mg/L, and the condition at the Ni concentration of 62.5 mg/L when the Feconcentration was at its lowest (32.5 mg/L).An analysis of variance was performed for the factorial design with Minitabsoftware setting H2 productivity as the response and Ni concentration, Fe concentration,and the combination of Ni and Fe concentrations as the factors (Table 6). For this1.2-J IO.8O.6U2 0.4(.4I0.20Fe IeeI: 0; Fe Ieel: I; Fe leel: 2; Fe level: 0; Fe Ieel: I; Fe Ieel: 2; Fe leel: 0; Fe level: I; Fe Ieel: 2;Ni leeI: 0 Ni leel: 0 Ni Ieel: 0 Ni leeI: I Ni leel: I Ni leeI: I Ni Ie’el: 2 Ni leeI: 2 Ni leeI: 256analysis, the H2 productivity was calculated as the slope of H2 concentration versus timebetween 10 and 48 hours. Nickel was shown to have thestrongest effect with the smallestP-value of 0.194, even though this effect was notstatistically significant at a 95%confidence level. Both the effect of Fe and theinteraction effect between Ni and Fe werenot detected at a confidence level of 95%. Althoughthe ANOVA did not detect anysignificant effects, the plot of main effects generated bythe Minitab software showed alinear increase in H2 productivity with increasing Nickel concentration, and adecrease inH2 productivity at the highest level of Fe (Figure 16).Table 6- Analysis of variance (ANOVA) for the effect of Fe and Ni on H2 productivity between 10and 48 hours.Source OF SegSS AdjSS AdjMS F PNi level 2 0.047795 0.030218 0.015109 1.81 0.194Fe level 2 0.008734 0.008645 0.004323 0.52 0.605NiIevel*Felevel 4 0.018312 0.018312 0.004578 0.55 0.703Error 17 0.141938 0.141938 0.008349Total 25 0.216779/.////Figure 16 - Effects of nickel and iron on H2 productivity between 10 and 48 hours. Graph generatedby Minitab software during ANOVA analysis.Ni levelMain Effects Plot (fitted means) for H2 productivityFe level0.6500.6250.6000.575-0.550-II0.0 62.5 125.0 32.5 125.0 250.057Another ANOVA analysis was performed with Minitab using H2 productivity asthe response, but this time using only Ni as a factor and ignoring any effect of Fe. WhileFe was not considered as a factor, all of the data collected from the factorial design wasstill used for the analysis. The results show a P-value of 0.057 for the effect of Niconcentration (Table 7). This analysis suggests Ni did have a statistically significantimpact on hydrogen productivity at a confidence level of 94%. In order to visualize theeffect of Ni concentration on both H2 productivity and time at which the maximumproductivity was attained, H2 productivity was plotted versus time, and a bar graph of theH2 productivities used for the statistical analysis is also shown (Figures 17 and 18). TheH2 productivity used for the statistical analysis was the slope of 112 concentration versustime graph between 10 and 48 hours. Note that the H2 productivity only decreased afterattaining a maximum because the experiments were performed in batch reactors, so thesubstrate, CO, was depleted.Table 7- Analysis of variance for the effect of Ni on H2 productivity between 10 and 48 hours.Source DF Seq SS Adj SS Adj MS F PNi level 2 0.047795 0.047795 0.023897 325 0.057Error 23 0.168985 0.168985 0.007347rrotal25 0.216779Figure 17 demonstrates an almost two-fold increase in H2 productivity when theconcentration of Ni was increased from 0 to 125 mg/L at a time of 20 hours, when Fe wasat level 0. A hypothesis test on the mean of the overall productivity between 10 and 48hours for both of these Ni concentrations showed a statistically significant increase ofvolumetric H2 productivity at a 95% confidence level. Also, the maximum H2productivity was reached at a notably shorter time for increased Ni concentrations,suggesting that the active enzyme complex was present sooner (Figure 18). These resultssuggest that CO-dehydrogenase and hydrogenase do bind to nickel and its presenceincreases the efficiency of enzyme activation, as well as the 112 productivity.581.41010.8E0.6>4-C,0.0aa.x-J0E>U2c•4I1.2-0.20 20 40 60 80 100Time (hours)Figure 17 - The effect of nickel concentration on the evolution of H2 productivity throughout thebatch H2 production phase when Iron concentration is kept constant at 32.Smg/L. Symbols: -+-,OmgIL Ni; -s-, 62.5mg/L Ni; -. A , l25mgfL Ni. Data points represent the average of 4 runs and theerror bars represent the standard deviation.ax=2551S0.90.80.70.60.50.40.30.20.10.0Omg/LNI 62.5 mg/L Ni 125 mg/L NiFigure 18 - Effect of nickel on the H2 productivity between 10 and 48 hours, and the timeat whichthe maximum productivity is reached when Fe is present at the constant level of 32.5 mgIL. Data barsrepresent the average of four measurements and the error bars represent the standard deviations.59The effect of iron on H2 productivity between 10 and 48 hourswhen no nickelwas present is shown in Figure 19. The data suggests thatthere was no effect onproductivity when increasing iron concentration from32.5 mg/L to 250 mg/L. There wasalso a slight increase in time at which the maximumproductivity was reached whenincreasing the concentration. The interaction effect shownin Figure 15 was re-iterated inFigure 20, a graph generated by Minitab duringthe full factorial analysis. Theoverlapping curves in this figure demonstrate that therewas an interaction effect betweenNi and Fe at nickel concentrations of 0 and 62.5mg/L.Figure 20 also demonstrates thetrend of decreasing H2 productivity for increasingFe levels at higher nickelconcentrations. Together, this implies that the lowconcentration of iron available in thePTMI salt solution was sufficient for the activationof the enzymes used during H2production. Increasing the concentration of Feabove 32.5mg/L is not necessary andcould be detrimental to the timing of the process,as well as the productivity when nickelis available in the media.0.7O.6x0=0.5-j0E 0.44-’0.2_____xO.l0.032.5 mg/L Fe 125 mg/L Fe 250mg/L FeFigure 19 - Effect of iron on H2 productivity between 10and 48 hours, and the time at which themaximum H2 productivity is reached when no Ni is present.Data bars represent the average of 4measurements at 32.5 mg/L and the average of 2 measurementsat 125 and 250mg/L. Error barsrepresent standard deviation.tmax33.3 hrstnlax34.0 hrstmax36.3 hrsii60Figure 20 - The interaction effect of Ni and Fe on H2 productivity. Graph generatedby Minitabsoftware during ANOVA analysis.5.5 Strategyfor minimizing inhibition ofenzymeproductionRe-suspension was a process performed between the aerobic growth andanaerobic H2 production stages. The broth was spun down after growthand the spentmedia discarded. The cells were then re-suspended in fresh media.This process allowedfor any inhibitory metabolites that were produced during the growth stageto bediscarded. Re-suspension also allowed cell density to be increasedby combining severalbottles from growth into one for H2 production. While increasingcell density will bediscussed in a later section, this section focuses onthe effect of discarding the spentmedia, and replacing it with fresh media of the same initial composition.The results indicate that re-suspension significantly decreased thelag phase, andslightly increased the H2 productivity (Figure 21). TheH2 productivities were measuredas the slope of consecutive data points on the H2 concentrationcurves during the H2production phase, disregarding the lag and stationary phases. The volumetricH2productivities were approximately 0.95 and 1.25 mmol H2! (L x h) forthe case withoutre-suspension and with re-suspension, respectively. The specificH2 productivities0.70Ni level—.-—- 0.0••• 62,5• i9_j40.65C0.600.5532.5a125.0 250.0Fe level61followed a similar trend to the volumetric productivities at 0.31 and 0.53 mmol 112/ (gcell x h) for the case without re-suspension and with re-suspension, respectively. The lagphase decreased from approximately 18 hours to 5 hours with re-suspension. Theseresults suggest that some inhibitory metabolites are being produced during the aerobicgrowth stage. The metabolites produced, directly orindirectly, are inhibiting 112production by decreasing the H2 productivity, but theyare also inhibiting the productionor activation of the CO dehydrogenase and hydrogenase enzymes.40.0—35.0______________-----30.025.020.015.0_______________________—+—No re-suspension ———Re-suspension in fresh media0U10.0I’1I5.00.0I0 20 40 60 80 100Time (hours)Figure 21 - The effect of re-suspending the cells between the growth and H2 production phases on H2concentration evolution. . Symbols: —•—, cells are not re-suspended; —-, cells are re-suspendedbetween the growth and H2 production phases. Data points represent the average of 2 runs and theerror bars represent the standard deviation.Although the exact nature of these inhibitory metabolites is unknown, the HPLCresults in Figure 6 show that both lactate and acetate were produced during growth. Thesesmall organic acids, present after open growth conditions with no buffer at concentrationsof 4 g/L lactic acid and 2.3 g/L acetic acid, could be inhibiting the enzyme activity.Typical inhibition levels of lacate and acetate on hydrogenase and CO dehydrogenasecould not be found in the literature but are assumed to be below the 40mM concentrationobserved for both lactate and acetate after the growth stage. While the organic acids maybe inhibitory themselves at these concentrations, it is also conceivable that it is the62decrease in pH that these organicacids cause that inhibits the enzymes. As seen in section5.3, Ci amalonaticus Y]9 is verysensitive to pH, so it should be monitored, particularlyunder conditions that produce organicacids.5.6 Mass transferDue to the low solubility of CO,external mass transfer of the substrate CO fromthe gas phase into the liquid phasehas been one of the major obstacles reported for theuse of the biological water-gas shift reaction (Amos2004, Wolfrum, Watt & Huang2002, Maness, Weaver 2002, Woifrum, Watt2001). Several solutions have beensuggested to solve this mass transfer obstacle usingnovel reactor designs, but beforeinvestigating complex reactor designs,studies were performed to see how basicproperties such as pressure and cell density affected H2productivity.On a theoretical basis, two solutions existfor resolving external mass transferlimitations: increasing the mass transfer coefficient (kLa)or increasing the substratesolubility. Increasing mixing and increasing the areaavailable for mass transfer wouldboth increase the mass transfer coefficient. Agitatingthe reactor more vigorously canincrease mixing, while using small gas bubbles withhigh surface to volume ratio and lowrise-velocities can increase area. Since the systemis already being agitated in a shakingincubator at a high speed of 250 rpm, and the systemis batch so the bubble size cannot becontrolled, the substrate solubility was investigatedrather than the mass transfercoefficient.The solubility of CO in water isapproximately 0.85 mmol/L at atmosphericpressure and 30°C. Decreasing the temperatureor increasing the CO partial pressure, asseen in Henry’s law, can increase this low solubility:C0 kHpco(16)where Cco represents the concentration of CO insolution (M), kH is Henry’s law constant(M/atm), andpcois the partial pressure of CO above the solution (atm). Henry’slawconstant for CO, at atmospheric temperature, is9.5 x i04 M/atm. Increasing the COpartial pressure should thus proportionately increase CO concentration in theliquidphase; therefore, if the system is mass transfer limited, this should proportionately63._ 50.0C.-J:30.020.00z10.00.0Jziat71—U—1.5atm:—*—2atm0 20 40 60 80 100Time (hours)Figure 22 - The effect of pressure on H2 concentration. Symbols: —+—, 1 atm; —a—, 1.5 atm;- A , 2atm. Data points represent the average of 2 runs and the error bars represent the standarddeviation.The line connects the data points for ease of visualisation.increase H2 productivity. Batch experiments wereperformed at increased CO partialpressure to test this theory.While many initial attempts failed at accurately measuringthe effect of increasingCO partial pressure, an experiment was designedthat was accurate and precise. In thisexperiment, the pressure was increased with the pre-mixed40% CO in He (v/v) gascylinder and monitored with a portable pressure gauge. The pressuregauge wasconnected to a needle for ease of measurements through thesepta. The results of theseexperiments showed that increasing the pressure from 1 atmosphere (atm) to1.5 atm hadno significant effect on H2 productivity, and increasingto 2 atm decreased H2productivity significantly (Figures 22 and 23). Note that the time evolutionof H2concentration in Figure 22 is not accurately illustrated because few datapoints weretaken to prevent depressurizing the reactors. The results do not follow the expectedtrendof proportional increase of H2 productivity with increased pressure, but rather show theopposite effect at high pressures. This implies substrate inhibition at increased CO partialpressures.60.0640.7Figure 23 - The effect of pressure onmaximum H2 productivity. Databars represent the averageof 2runs and the error bars represent thestandard deviation.Substrate inhibition istaking place because ofthe higher availability of COsubstrate dissolved inthe liquid phase at higherpressures. This behavior athigh pressuresis analogous to the behaviorof the cells when the bottleswere purged with pure CObefore the concentrationwas adjusted to the desiredinitial CO concentration(Figure 11).Both of these results suggestsubstrate inhibition at veryhigh CO concentrationsin theliquid phase. Anotherpossible explanation forthe decreased productivityat 2atmospheres is that this particularstrain of bacterial cellsmay not be able to surviveunder these environmentalconditions. Water, however,is an incompressible fluid,so thisis highly unlikely becausein this solvent, the cells will notknow the difference between1 atm and 2atm. Overall,these results suggest thatincreasing the pressure inthe systemdoes not resolve any possiblemass transfer limitationbecause of the substrateinhibitionthat comes into play athigh pressure. Alternatively,the results could mean thatmasstransfer limitation is nota major and significant issue forthis particular organism,butrather the focus should beon alleviating the inhibition at highsubstrate concentrations.0.6x0.5-jO.4p0.3>1eDl0.10.00.21.0 atm, 40%CO1.5 atm, 40%CO 2.0atm, 40%CO655.7 Effect ofcell concentrationSince mass transfer was not limiting H2productivity, increasing the densityofcells in the reactor was investigated to increaseH2 production. Increasing the celldensityshould increase the volumetric H2 productivityproportionally. An initial experimenttotest this theory was performedby decreasing cell density by dilution withfresh mediaafter the growth stage to 75% and 50% ofthe initial cell density. This initialtest gave theopposite of the expected result,by having very similar H2 productivitiesfor all cellconcentrations rather than a decreasingvolumetric productivityfor decreasing celldensity (Appendix B). The specificH2 productivity thus increased withdecreasing celldensity. This result could have been explainedby a fast growth rate of the cells,equalizing cell density back to approximately2.5 g/L while the reactors werebeingswitched into the anaerobic H2 productionstage, but this explanation was nullifiedwhencell density was measured beforeand after the anaerobic stage andthe ratios were foundto remain constant.A more accurate test on the effect of celldensity was effectuated by spinningdown the cells after growth from four serumbottles and combining these intoone serumbottle, thus quadrupling the cell densitybefore the start of the anaerobic stage.The timeevolution of H2 concentration, and acomparison of volumetric andspecific H2productivities are illustratedfor this experiment in Figures 24 and25, respectively. Theseresults show that quadrupling the celldensity gave very similar H2 productioncurves,which translates into no significant differencein volumetric H2 productivity,but muchlower specific H2 productivity.The specific H2 productivity decreasedfrom 0.5 to 0.1mmol H2 /(g cell x h) when the cell density was quadrupled.66404-C.).20.(.4I35-j[25.2 2015C0110500 20 40 60 80 100Time (hours)Figure 24 - The effect of quadrupling cell density on H2 concentration. Symbols: -4-, regular celldensity; —I—, 4 times higher cell density. Data points represent the average of 2 runs and the errorbars represent the standard deviation.Maxvolumetric H2 productivity (mmol/(L liq xh))• Max specific H2 productivity (mmol/ (g cell x h))1.61.41.21.00.80.60.40.20.0Resuspension Resuspension and celldensity*4Figure 25 - The effect of quadrupling cell density on maximum hydrogen productivity. Data barsrepresent the average of 2 runs and the error bars represent the standard deviation.67This result was troublingbecause everythingin biotechnologypoints toward anexpected proportional increasein H2 production when celldensity is increased.Thespecific productivity ofthe cells should notchange with cell density.One possibleexplanation for this is thatthe system undergoes productinhibition. If theinitial enzymeload were increased, the H2would be generated ata faster rate. Ifhigh H2 concentrationsinhibit the enzymes,then this could explainwhy the evolutionof H2 concentrationdoesnot change when thecell density is increased.Since there is no H2at the start of theanaerobic stage, this effectwould only be initiatedafter the H2 concentrationreaches acertain point. This wasnot the case, as theinitial H2 productivitywas not increased atanincreased cell density.Another explanationwould be that the cellssomehow regulatethemselves, andthere is a limit on theviable cell count. Ifthe number of viablecells is not proportionaltothe total cell densitybut rather stays constanteven when increasingthe total numberofcells, and only theviable cells produceH2, then this wouldexplain the constantvolumetric H2 productivity.Perhaps the cell turnoveris a lot faster thanthe H2 productionprocess, allowingthe cells to regulatethemselves toa maximum viability level.Performing viable cellassays rather than simplyusing optical densityto measure celldensity could verifythis theory. Anotherform of cell regulationis quorum sensing. Somebacteria use quorumsensing to regulate certainbehaviors basedon the local celldensityof the bacterial cells. Quorumsensing is a form ofcommunication amongbacteria wheresignaling moleculesare secreted and, whenbound to receptors,activate the transcriptionof certain genes. Thisprocess would needto be investigatedfurther to confirmthepresence of quorumsensing in Ci amalonaticusY19.A third possible reasonfor this unusual resultis that the enzymeconcentration isso high that adding moreenzymes does not affectthe conversion kinetics.If this were thecase, the productivitywould only increasewith an increasein specific activityof theenzyme, not withenzyme load. In orderto increase specificactivity, a more in depthlookinside the cell wouldbe necessary to understandthe mechanismof CO conversionandthe enzymatic processes.Once this mechanismwas understood,genetic modificationscould be performedto increase thespecific enzyme activity,thus having morecontrolover H2 productivity.685.8 Prospects and limitations ofH2productivityThe maximum volumetric H2 productivityobtained was 1.5 mmol H2 / (L x h) fora system buffered to pH 6.5 withre-suspension between the growth and H2 productionstages. To find out the limitation or prospect of thisrate of H2 production, it must be putinto the context of a practical application.While several practical applications exist, onlyone has been studied and publishedfor the biological H2 production system by Levin etal. (2004).In the study by Levin et al, the size ofbio-hydrogen systems required to powerproton exchange membrane fuel cells (PEMFC) of varioussizes was calculated based onhydrogen production rates (Levin, Pitt & Love2004). Unpublished data by BC hydrosuggests that the average non-electrically heatedhouse in British Columbia usesapproximately 13,000 kWh electricity every year.This amount of energy would require aPEMFC of 1.5 kW output power, assumingit runs at constant output, thus ignoring thediurnal and seasonal variations. Using the conversionfactors from this research paper andthe maximum volumetric productivity of 1.5 mmolH2 / (L x h), the bioreactor size wascalculated. The result was that a 23,900L bioreactor would berequired to supplysufficient H2 to power a PEMFC of 1.5 kW using the biologicalwater gas shift reactioncatalyzed by Ci amalonaticus Y19. This bioreactorsize is enormous, making thistechnology economically unfeasible for this application.Productivities at least 100 timeshigher must be achieved to consider this technologyapplication.Another possible application of this technologywould be to rid a gas stream ofCO, which can be considered a toxic impurity, andthe H2 production could be seen as aside benefit. On top of this, low-grade synthesis gasis considered a waste stream, and anyenergy recovered from this stream can result inpositive gain. In order to assess thefeasibility of these practical applications, economicanalyses would need to be performedon producing H2 or getting rid of CO from a gasstream.696.0 ConclusionsIn this research project, a two-step reactor protocol was successfully developedusing Ci amalonaticus Y19 to catalyze the biological WGS reaction. While this systemwas not fully optimized, it can serve as a reference point for further work in this field.The most important result found from the investigation of the effect of mediumcomponents was the significant improvements to both the volumetric H2 productivity andthe time of activation of CO dehydrogenase and CO-induced hydrogenase when the LBmedia was supplemented with nickel at a concentration of 125 mg/L. Other mediacomponents investigated were the presence of CO during the growth stage, the additionof other carbon sources, and the deletion of tryptone. The results indicated that the sealedgrowth condition with CO present was not essential to H2 production in the followinganaerobic stage, and actually decreased the H2 productivity. Open growth conditions arethus not only preferred, but also greatly simplifi the growth protocol. The addition ofexternal carbon sources ceased 112 production by introducing a preferred substrate for Ciamalonaticus Y19 that induced anaerobic cell growth rather than conversion of CO.Tryptone in the media was deemed to act as a buffer but the deletion of tryptone did notincrease H2 productivity as was predicted in a previous study on Ci amalonaticus Y19. Itwas found that H2 production did not rely on the presence of tryptone, meaning Ciamalonaticus Y19 was not auxotrophic for the amino acids contained in the enzymaticdigest of casein.Process conditions investigated included pH, re-suspension, pressure, and celldensity. H2 production by Ci amalonaticus Y19 was strongly pH sensitive and improvedwith pH control. It was observed that a pH greater than 5.5 was indispensable to theproduction of H2. Re-suspension between the aerobic growth and anaerobic H2production stages decreased the inhibition of enzyme production and activation byeliminating the inhibitory metabolites produced during the growth stage. The expectedmass transfer limitation, due to the low solubility of the gaseous substrate, CO, in thewater gas shift reaction, was not observed, and increasing the partial pressure of thesubstrate had a negative effect at pressures above 2 atmospheres. This decrease in H2productivity at higher pressures is likely due to substrate inhibition compensating70increased substrate availability. The attempt at increasingthe cell density also gavedisconcerting results that showed no improvement in volumetric H2productivity atincreased cell densities.Overall, this research project has shown that Ci amalonaticusY19 offers moderateCO uptake rates and conversion yields close to theoreticalmaximum. 112 production bythe biological water gas shift reaction catalyzed by Ciamalonaticus Y19 was stronglyinfluenced by the media composition and the pH. An initial optimizationof the processled to an open growth phase, followed by re-suspension ofthe media between phases, andan addition of nickel to LB media for the anaerobic stage. The pH must also be controlledthroughout this process.717.0 RecommendationsThroughout this research project,insight was given as tohow several mediumcomponents, as well as howpressure and other process parametersaffect the H2productivity of Ci amalonaticusY19 catalyzing the biological watergas shift reaction(bio-WGS). The maximum volumetricH2 productivity obtained was 1.5mmol 112/ (L xh) for a buffered system withre-suspension between the growthand H2 productionstages. This H2 production ratewas found to be economicallyunviable for sometechnological applications such as providingelectrical power to a housethrough a 1.5kWproton exchange membrane fuelcell (PEMFC).One recommendation for furtherresearch is to calculate theeconomic viability ofthis 112 production rate for other practicalapplications. These applicationcould include asmaller scale process where anyenergy recovered from the low-gradesyngas wastestream can result in positive gain.Also, this process couldbe used as a method to ridagas stream of CO, which can beconsidered a toxic impurity,and the H2 production couldbe seen as a side benefit.The experiment where cell densitywas quadrupled and the volumetricrate of H2production was not increased remainsa mystery. Solving this mysterywould beparticularly interesting, and couldoptimistically be accomplishedby doing either viablecell assays, or looking inside thecell to learn more about the enzymekinetics. Creatingassays for the enzymes wouldhelp to learn more about what stagein the process theenzymes are produced and howmuch enzyme is produced. Findingthe sequences of theenzymes would also be informative.Once the enzyme kinetics areunderstood, geneticmanipulations could increase thespecific activity of the keyenzymes of the biologicalwater gas shift reactions:CO dehydrogenase, and CO-inducedhydrogenase.A more in depth optimizationof the media compositionis also recommended.This would decrease the possibilityof any unknown componentsin the complex mediaaffecting the outcome of H2production. 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Available:http://biodiesel.environmentalactiongroup.org/hydrogen.htm1[2008, September/23].Winkler, M., Hemschemeier,A., Gotor, C., Melis, A. & Happe,T. 2002, “[Fe]hydrogenases in green algae:photo-fermentation and hydrogenevolution under sulfurdeprivation”, International JournalofHydrogen Energy, vol. 27,no. 11-12,pp.143 1-1439.78Woifrum, E.J. & Watt, A.S. 2001,Bioreactor design studiesfor anovel hydrogen-producing bacterium, NationalRenewable Energy Laboratory,Golden, CO. NREL/CP570-30535Woifrum, E.J., Watt, A.S. &Huang, 1. 2002, Bioreactor developmentfor biologicalhydrogen production, NationalRenewable Energy Laboratory, Golden, CO.NREL/CP610-3240579AppendicesAppendix A — Calibration curvesGlycerol stocksGlycerol stock solutions of Citrobacteramalonaticus 179 were made from theagar shipments from Korea. In a l25mL erlenmeyerflask, 4OmL of the LB-PTM1 media(Tables 3 and 4) was inoculated with 0.3mL of Ciamalonaticus Y19 in agar. The flaskwas incubated at 30°C and 250rpm,and the optical density was measured every houruntil it reached approximately 1.5.Pure glycerol was added until a final concentration of20% glycerol was reached. Thesolution was stored in lmL samples in eppendorf tubesat—80°C.GC calibration curvesExternal calibration was performed for H2,CO andCO2in order to convert the GC areaoutput into concentrations.I2016128400.00 0.10 0.20 0.30 0.40 0.50112concentration (mM)0.60 0.70 0.80 0.90Figure 26 - Hydrogen calibration performed at a pressureof 5psi on May23rd,2007804036322821.38x= 0.998924128400.00 0.20 0.40 0.60 0.80 1.001.20 1.40 1.60 1.80H2 concentration (mM)Figure 27 - Hydrogen calibration performed at apressure of 5psi on April22m1,20084000350030002500y = 84.825xR2=0.995520001500100050000.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.0045.00CO concentration (mM)Figure 28— Carbon monoxide calibration performed at a pressure of lipsi on June29tb,200781450040003500y= 96.662x30002- R =0.99842500220001500100050000.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00CO concentration (mMFigure 29 - Carbon monoxide calibration performed at a pressure of Spsi on November27th2007350030002500y = 97.3 83xR2=0.9993200021500100050000.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00CO concentration (mM)Figure 30 - Carbon monoxide calibration performed at a pressure of 5psi on April 200882100908070y=46.222x60R2=O.9945!50403020100 I I I I0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.41.6 1.8 2.0C02 concentration (mM)Figure 31 - Carbon dioxide calibration performed at a pressureof lipsi on October 29th, 2007100908070‘ 60y=47.176xR2=09984E4030201000.0 0.2 0.4 0.6 0.8 1.0 1.2 1.41.6 1.8 2.0C02 concentration (mM)Figure 32 - Carbon dioxide calibration performed at a pressureof 5psi on November 28th, 20078310090807048.874x60R2=O.999540-3020100-0.0 0.5 1.0 1.5 2.0C02 concentration (mM)Figure 33 - Carbon dioxide calibration performed at a pressure of 5psion April 23rd, 2008HPLC calibration curvesExternal calibration was performed for acetate and lactate in order to converttheHPLC area output into concentrations.84140000012000001::::::<600000= 59326x::::::R2=120 25Acetate concentration (gIL)Figure 34 - Acetate calibration performedon the HPLC on July30tI,200818000001600000T1400000y = 70992xR2=1j1200000100000080000060000040000020000000 5 1015 20 25Lactate concentration (giL)Figure 35 - Lactate calibration performedon the HPLC on July 30th, 200885Appendix B — H2 concentrationdataLogarithmic growth curveFigure 36- Logarithmic Citrobacter amalonaticus Y19growth curve under two conditions. Symbols: -•-, cell concentration under completelyaerobic conditions; -•-, cell concentration under sealedaerobic conditions with 8% CO (v/v) present in the headspace.Symbols represent data points, andlines connect the symbols for ease of visualization.• Effect of CO concentrationsFigure 37 - Batch evolution of H2 concentration forvarying initial CO concentrations. COconcentrations vary from 7 to 60% CO inhelium (vlv). Symbols: -i-, 7% CO; -.-, 14% CO; -A-,27% CO; -.-, 40% CO; 60% CO. Symbols representthe experimental data and lines representthe3tddegree polynomial best-fit curves.654‘32100 2 4 6 8Time (hours)10 12 144035-j25E20015010045I0-50 20 40 60 80100Time (hours)8640—e—40% CO (He purge)—-- 60% CO (He purge)-*-40%CO(COpurge)—.--60% CO (CO purge)—*-- 80% CO (COpurge)—0—100% CO (CO purge)..Figure 38 - Effect of the purging gas used (COor He) on the H2 concentration evolution. InitialCOconcentration adjusted after purge from 40-100%(v/v). Symbols: -+-, 40% CO, purged with He; -i-60% CO, purged with He;- A-, 40% CO, purged with CO; -.-, 60% CO, purged with CO; 80%CO, purged with CO; -0-, 100% CO,purged with CO. Symbols represent data points, and linesconnect the symbols for easeof visualization.. Effect of buffer4012520Ca 15C)C010IFigure 39 - Effect of adding 20mMphosphate buffer to several conditions. Symbols:-+-, LB, nobuffer; -.-, LB, buffered; -A-,tryptone deleted LB, no buffer; -.-, tryptone deletedLB, buffered;--CO sealed, no buffer;-I-,CO sealed, buffered. Symbols represent average of 2experimental runsand error bars represent the standarddeviation. Lines connect the symbols for ease of visualization35:3042520Ca 15C010I500 2040 60 80 100120Time (hours)35—‘—e—LB growth, re-suspensionin LB—.—LB growth, re-suspension in LB, with buffer—fr—LB growth, resuspension in Tryp-del LB——.—LB growth, resuspension in Tryp-del LB, withbuffer—*—Growth sealed with CO——i-—Growth sealed with CO, with buffer• A. .fr$500 20 40 6080 100Time (hours)120’870•I0EEC0IFigure 40- Effect of Fe concentration on H2 concentration when no Ni present. Symbols: -4-,32.5mgfL Fe; -.-, l25mgfL Fe; -A-, 25OmgfL Fe. Symbols represent average of experimental runsand error bars represent the standard deviation. Lines connect the symbols for ease of visualization.}/ —— Fe: 0, Ni: 0—--Fe:0,Ni:1—--Fe: 0, Ni: 2Figure 41- Effect of Ni concentration on H2 concentration when 32.5mg/L Fe present. Symbols: -4-,Omg/L Ni; -.-, 62.5mg/L Ni; -A-, 125mg/L Ni. Symbols represent average of experimental runs anderror bars represent the standard deviation. Lines connect the symbols for ease of visualization.Effect of Ni and Fe4035302520151050——Fe:0, NE47 —--Fe:1,Ni:0--A—Fe:2,Ni:00 20 40 60 80 100Time (hours)4035.J3O25_______ ______ ______________20_______C0____________ ___________________________________________(.) 10I50 I I I0 20 40 60 80 100Time (hours)88l.4j1.293fr-31.5 hrs. 34.0 hrs. It,,,,, =36.335.0 hrs 33.029.0 hrs0.6c.=.eo.4Q.e4I0.20Figure 42 - Maximum H2 productivity for all the combinations of levelsin the factorial design on Feand Ni concentrations. Data bars represent the average of 2or 4 runs depending on the condition,and the error bars represent the standard deviation. The averagetime at which the maximumproductivity is attained is shown above the data bars.60 80 100Figure 43- Effect of glucose on112 concentration. Symbols: -i-, No glucose; -•-, Glucose in H2production phase; -Arn, Glucose during growth andH2 production. Symbols represent average ofexperimental runs and error bars represent the standard deviation.Lines connect the symbols forease of visualization.25.5 hrsjFe leeI: 0; Fe level: 1; Fe leel: 2; Fe leel: 0; Fe level: 1; Fe level: 2; Fe leel: 0; Fe le\el:1; Fe leel: 2;Ni leel: 0 Ni lel: 0 Ni Ieel: 0 Ni Ieel: 1 Ni Ieel: 1 Ni level: 1Ni eel: 2 Ni leel: 2 Ni leeI: 2. Effect of adding glucose740350•3025.2 201510I50—4—LB media with re-suspension—— LB growth, re-suspended in LB and GlucoseH—A-- LB & Glucose for growth and after re-suspension0 20 40Time (hours)89• Effect of deleting tryptone4035-f-J0E 25E.2 2015 —4—LBgrowth, re-suspension in LBg ——LB growth, re-suspension in LB, with bufferic—*LB growth, resuspension in Tryp-del LB——LB growth, resuspension in Tryp-del LB, with buffer50 A -m r— A -10 20 40 60 80 100120Time (hours)Figure 44- Effect of tryptone deletion, with and without 20mMphosphate buffer. Symbols: -+-, LB,no buffer; -.-, LII, buffered; - A-, Tryptone deleted LII, no buffer; -.-, Tryptonedeleted LB,buffered. Symbols represent average of 2 experimental runs and error bars representthe standarddeviation. Lines connect the symbols for ease of visualization• Effect of CO during growth____ILB growth, re-suspension in LB........Growth sealed with CC, no resuspension—*--Growth sealed with CO, buffered, no resuspension—-—Growth sealed with CC, resuspension in LB0 20 40 60 80100 120Time (hours)Figure 45- Effect of sealing the growth with 6.25mL CO tothe headspace under several conditions.Symbols: -+-, LB growth, re-suspended in LB; -a-, Sealed LBgrowth with CO; - A , Sealed LBgrowth with CO, buffered; -.-, Sealed LB growth with CO,re-suspended in LB. Symbols representaverage of 2 experimental runs and error bars represent thestandard deviation. Lines connect thesymbols for ease of visualization5045- 403543025200C00Nx 105090. Effect of re-suspension40 60 80 100Time (hours)Figure 46 - The effect of re-suspendingthe cells between the growth and H2production phases on H2concentration evolution.. Symbols: -i-, cells are not re-suspended;-u-, cells are re-suspendedbetween the growth and H2production phases. Data points representthe average of 2 runs and theerror bars represent the standard deviation.. Effect of increasing CO partialpressure-j0E:30.04-20.00C.)C”IFigure 47-The effect of pressure on H2concentration. Symbols: -i-, 1 atm; -.-, 1.5 atm;-A, 2 atm.Data points represent the average of2 runs and the error bars represent the standarddeviation.40.035.030.025.0C.-J0EE20.0.1w 15.0C.)0C.)10.0I5.0/• No re-suspension—— Re-suspension in fresh media0.00 2060.050.040.0—j1 atm—m--1.5 atm—j--2atm—10.00.00 20 4060 80 100Time (hours)91-J0Ei04-4-a)C.)0U(NI. Effect of decreasing cell concentration0-J0EEC0I121086420//—4—100% [celij],//1[cell]/J/—k— 50% [cell]“1/ii,//I I I0 10 20 30 40 5060 70 80Time (hours)Figure 48- Effect of dilution (decreased cell density) on H2concentration. Symbols: -i-, no dilution,100% cell concentration; -i-, 75% cell concentration; A , 50%cell concentration. Points representthe experimental data and lines connect the symbols for easeof visualization. Effect of increasing cell concentration403530252015105—a-- Re-suspension.--. Re-suspension and celldensity*40 20 40Time (hours)060 80 100Figure 49-The effect of quadrupling cell density on 112 concentration. Symbols: -+-, regular celldensity; -.-, 4 times higher cell density. Data points represent the average of 2 runs and the errorbars represent the standard deviation.92. Max conditions1z-tJ,,/—4-—Noresuspension—.—Resuspension in LB—*— Resuspensionin LB, with buffer• Resuspension in LB, with Nickel—*-- Resuspensionin LB, with buffer and NickelFigure 50- H2 concentration profile ofmaximum conditions. Symbols: -+-, LB; -s, LB,re-suspended;- A —, LB, re-suspended and buffered;-.-, LB, re-suspended and Ni added;-,LB, re-suspended,buffered, and Ni added. Symbols representaverage of 2 experimental runs and error barsrepresentthe standard deviation. Lines connectthe symbols for ease of visualization.40350•30E252O-ICa,C.,C0C.’10500 2040 60 80Time (hours)100 12093

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