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Effects of calcium on anaerobic acidogenic biofilms Huang, Jifei 1994

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EFFECTS OF CALCIUM ON ANAEROBICACIDOGENIC BIOFILMSbyJifei HuangB.Eng., South China Institute of Technology, 1982A THESIS SUBMITTED iN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFMASTER OF APPLIED SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMICAL ENGINEERINGWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMARCH, 1994© Jifei Huang, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________Department of CkLThe University of British ColumbiaVancouver, CanadaDate ‘“‘ ‘C’ ) i 94DE-6 (2/88)ABSTRACTCalcium has been found to be involved in formation and development of thebioflims for many species of bacteria, but effects of calcium on anaerobic bioflims forindustrial application have rarely been reported. In this study, a mixed-culture of anaerobicbacterial bioflims were grown in lactose cultural medium with various calciumconcentrations -- 1.2, 80, 100, 120, 170 and 230 mg/I. Specially designed CSTR reactorswere used. The temperature and pH in the reactors were controlled at 35 °C and pH 4.5for optimal growth of acidogenic bacteria. The influence of calcium on biofllm dry mass,total organic carbon, immobilized calcium concentration and bioflim specific activity weremeasured.The biofllm mass accumulation was increased by the presence of calcium in thegrowth medium when calcium concentration was not higher than 120 mg/I. Calciumaccumulated in the bioflims increased in proportion to the calcium level in the feed. Thebioflims for an increased input calcium concentration showed a trend of decrease inspecific activity. The biofllms with a thickness of less than 0.5 mm had the highest specificactivity. The optimum calcium concentration for the substrate consumption by the biofilmswas 100 -120 mg/i.The bioflims transferred from higher calcium medium to lower calcium mediumwere more susceptible to sioughing from their support surfaces, indicating calcium’s rolein the stability of the bioflim structure.11TABLE OF CONTENTSPageAbstract.iiList of Tables viList of Figures viiAcknowledgment ixIntroduction 1Chapter 1 Literature Review 41.1 The structure and functions of bioflims 41.1.1 Thebioflimsystem 61.1.2 The functions of bioflim 71.1.2 The processes of bioflim formation and development 81.1.3 The factors affecting the biofilm processes 12Surface characteristics 13Nutrient concentration 14Cellular physiological status 15Fluid velocity 16pHofthefluid 17Temperature 17Presence of inorganic suspended particles 181.2 Biofilms for anaerobic wastewater treatment 181.2.1 Anaerobic digestion and related biofilm reactor technologies 19iii1.2.2 Characteristics of bioflims in anaerobic bioflim reactors 201.2.3 Modeling of anaerobic digestion in biofilm reactors 241.3 Calcium and bioflims 251.3.1 The bacterial roles of calcium 251.3.2 Previous studies in the effects of calcium on bacterial biofllms 28Chapter 2 Experimental 322.1 Experimental objectives 322.2 Experimental apparatus and conditions 332.2.1 Experimental set-up 332.2.2 Experimental conditions 352.2.3 Preparation of the bacteria inoculum 372.2.4 Start-up and maintenance of the reactors 392.3 Monitoring the reactor performance and Sampling 402.4 Sample analysis 412.4.1 Turbidity of bacterial suspension 422.4.2 Wet weight and dry weight of bioflims and suspended cells 422.4.3 Total organic carbon (TOC) 432.4.4 Lactose 432.4.5 Biofllm volume, density and thickness 452.4.6 Assay of bioflim specific activity 462.4.7 Total calcium 482.4.8 Biofilm minerals 482.4.9 Gaseous products 49Chapter 3 Results and Discussion 51iv3.1.Biofilmbiomass .513.2. Bioflim Thickness 563.3. Biofllm specific activity 633.4. Immobilized calcium 673.5 Influence of calcium fluctuation 733.6. Bioflim compositions and density 77Chapter 4 Conclusions and Recommendations 814.1 Conclusions 814.2 Recommendations 82Bibliography 85Appendix Experimental Data 94Bioflim TOC 94Dry Biofllm Mass 95Wet Bioflim Mass 96Average Ratio of TOCfbiofilm volume 97Bioflim Surface Area 98Bioflim Thickness and Volume 99Biofilm TOC Per Unit Area 100Bioflim Specific Activity Assay 102Cell and Substrate Concentration in Effluent 104Calcium within Bioflims 105VLIST OF TABLESPageTable 1 The Reactor Operating Conditions 36Table 2 The Composition of Culture Medium 37Table 3 The Calcium Concentrations in the Feed 38Table 4 The Composition of Bioflims 78viLIST OF FIGURESPageFigure 1 Illustration of the Experimental Set-up 34Figure 2 The Batch Reactor for Biofilm Specific Activity Assay 40Figure 3a Calibration Curve for Measurement Bacterial Solution Turbidity 44Figure 3b Standard Curve for Lactose Concentration Detennination 47Figure 3 A Typical Relation Curve of Cell Growth and Substrate Consumption forBioflim Specific Activity Assay 49Figure 4 Biofilm TOC as a Function of Time 52Figure 5 Biofilm Biomass as a Function of Calcium Concentration in Feed 54Figure 6 Biofilm Thickness as a Function of Time 57Figure 7 A Conceptual Device for Measurement ofBioflim Thickness 58Figure 8 Biofilm Carbon Concentration as a Function of Calcium Concentration in Feed62Figure 9 Bioflim Specific Activity as a Function of Calcium Concentration in Feed 64Figure 10 Biofilm Specific Activity as a Function of Time 65Figure 11 Bioflim Specific Activity as a Function of Bioflim Thickness 66Figure 12 Substrate Consumption Rate Per Unit Area Biofilm as a Function of CalciumConcentration in Feed 68Figure 13 Calcium Concentration within Bioflim as a Function of Calcium concentration inFeed 70Figure 14 Influence in Biomass Progression of Changes in Calcium Concentration duringBioflim Development 74Figure 15 Biomass Concentration in Effluent as a Function of Time 75, 76Figure 16 Biofilm Composition as a Function of Calcium Concentration in Feed 78viiFigure 17 Bioflim Density as a Function of Calcium Concentration in Feed 79Figure 18 An Alternative Design of the Experimental System for Study of AnaerobicBiofllms 83viiiACKNOWLEDGMENTI am very grateful for my supervisor, Dr. K.L. Pinder for his support, guidance andhelp in my completing this study.1 also want to thank Dr. Jian Yu and Dr. Rob Stephenson for their suggestions andhelp during the experiments. I appreciate Dr. R.M.R. Branion, Dr. S. Duff and Dr. K.V.Lo for their advice and review of this thesis.Special thanks go to my families for their continual support.ixINTRODUCTIONBacteria adhere to solid surfaces, grow and proliferate, and form a thin film on thesurfaces. This film consists of bacteria immobilized in the highly hydrated exopolymers andis called bioflim. It is a common form of life in bacterial ecology.Biofilm reactors that use bioflims as catalysts offer advantages over the traditionalsuspended cell reactors in maintaining a higher concentratiOn ofbacterial cells, which leadsto a faster reaction rate, and immobilization of the cells, which makes separation of thebiocatalysts and the desired products much easier. Biofilm reactors are also moreadvantageous than the other immobilized cell reactors in their simpler cell immobilizationprocesses, higher system stability and reduced diffusional resistance from the bulk liquid tothe immobilized cells. Because of their advantages, biofilm reactors have drawn a greatdeal of attention in the past ten years. Substantial advances have been made in thetechnology and industrial application of biofilm reactors, in particular, anaerobic bioflimreactors.Several types of anaerobic biofilm reactors have found their use in treatment ofwastewater from many industries, such as food processing, beverage, pulp and paperindustries, and agricultural wastes. Such advanced anaerobic digestion systems provide ahigher efficiency and stability in wastewater treatment operations than the traditionalanaerobic digestion systems. However, the major problems remain with these systems,including slow start-up, lack of control over the bioflim thickness or long term operationalstability, and difficulties in development of reliable models for scale up. This is not only1because of the nature of the anaerobic bacteria but also due to lack of understanding of themechanisms existing in the bioflim formation and development processes.In order to overcome these problems, many studies have been carried out on theinfluence of environmental factors, such as substrate concentration, pH, temperature andnutrients on performance of the anaerobic bioflim reactors. But little work has been doneso far on the effects of calcium on anaerobic bioflims for wastewater treatment, althoughcalcium is found to be important for the bioflims of many species of bacteria.Calcium is involved in bioflim formation and activities on three levels. First, on cell-environment; calcium may ‘condition’ the surfaces of support and bacterial cells. Forexample, calcium cations may alter the surface charge or electrostatic character of thesurfaces, thus facilitating bacterial adhesion to the support surface or bacterialaggregation. Second, on cell-cell; calcium plays a role in buildup of biofilm structures.Typically, calcium ions act as ‘cation bridges’ between polysaccharides originating fromdifferent cells. Third, within cells, calcium is required for certain biochemical reactions inbacteria and some bacterial physiological activities.Therefore, a study on the influence of calcium on anaerobic bioflims will bebeneficial for an understanding of anaerobic bioflim mechanisms and may be useful forcontrol of bioflim processes and practical operations in anaerobic wastewater treatment.This thesis includes five parts. In the first chapter, the general characteristics,structure and functions of biofilms, bioflim processes and influencing factors are outlined.Then the anaerobic biofilm characteristics and anaerobic digestion processes are brieflydiscussed. Finally, the previous work on calcium related to bioflims is reviewed. In thesecond chapter, the objectives and research scope of this study and the methodology usedin the experiments are introduced. The experimental results are presented and discussed inthe third chapter. The conclusions are given in the fourth chapter with some2recommendations for the further study. The raw experimental data related to the results inthe third chapter are attached in this thesis as an appendix.3CHAPTER 1LITERATURE REVIEWBiofilm formation and development comprises a series of processes resulting fromthe interactions between biological, chemical and physical parameters concerning theorganisms, the support material and the environment. An understanding of the structureand function of biofilms is essential for the prediction and control of biofilm processes.Research aimed at the understanding and predicting of biofllm structures and functions hasbeen advanced by scientists and engineers in many disciplines, in particular in generalmicrobiology, limnology, soil science, dental and medical sciences, and control ofindustrial biofouling and biocorrosion. Utilization of biofilms in up-to-date biologicalwastewater treatment technology has also contributed to the knowledge of bioflims andtheir mechanism. In this chapter, the general structure and function of a bioflim, bioflimprocesses and the factors influencing them will be first introduced. Then the studiesregarding properties of anaerobic bioflims important for waste water treatment will bereviewed. Finally, calcium’s role in bacteria and its effect on adhesion and bioflimdevelopment of bacteria, especially those used for anaerobic treatment of waste water, willbe discussed.41.1 The structure and functions of biofilmsBioflims consist mainly of water (79-95%), extracellular polymer substances BPS(70-95% of the organic matter of the dry bioflim mass), the microorganisms, and theembedded solid particles as well as dissolved substances (Flemming, Hans-Curt, 1993).Although the microorganisms account for only a minor part of the bioflim mass andvolume, they are the centre of the biological activities in bioflims. They excrete the EPSand control the physical and chemical properties of the bioflim. The BPS includepolysaccharides and glycoproteins. The mass of tangled fibres of polysaccharides orbranching sugar molecules extending from the bacterial surfaces forms the very adsorptiveand porous structure, or gel-like structure featured by biofilms in aquatic environments.Materials absorbed or entrapped in EPS include solute and inorganic particles - mostoften, calcium and magnesium. These materials play roles in the bioflim structure byinfluencing the growth and metabolism of cells, and/or are involved in bridging betweencells, polysaccharides and the substratum (Costerton, J.W., Geesey, G.G., & Cheng,K.J., 1978) or in maintenance of the tertiary structure of BPS (Turakhia, M.H., Cookey,K.E., & Characklis, W.G., 1983). The limited growth due to the diffusional transport ofnutrients from bulk liquid into the gel matrix is a major characteristics of bioflims. Thewater content of a biofilm is important for the enzyme activities and the transport ofnutrients and metabolites within the bioflims. Biofilms may be heterogenous in space andtime. The same bioflim can provide a variety of microenvironments for microbial systems.For example, in one-phase methanogenic biofilm reactors, the bioflim may contain anupper layer dominated by acidogenic bacteria and a base layer dominated by methanogenicbacteria. The bioflim surface is hydrophillic, rough, soft and ‘sticky’ (Characklis, W.G. &Marshall, K.C., 1990).51.1.1 The bioflim systemAccording to Wilderer and Characklis (Wilderer, P.A. & Characklis, W. G., 1989).a bioflim system includes five compartments: 1) the substratum, 2) the base film, 3) thesurface film, 4) the bulk liquid and 5) the gas. Each compartment can be described interms of its thermodynamic and transport properties as well as by the transport andtransformation processes that dominate within the compartment.The substratum plays a major role in biofilm processes during the early stages ofbiofilm accumulation and may influence the rate of cell accumulation as well as the initialcell population distribution. The base film consists of a rather structured microbiologyaccumulation, having relatively well-defined boundaries. Molecular (difii.isive) transportdominates in the base film. The surface film provides a transition between the bulk liquidcompartment and the base film. Gradients in biofilm properties in the direction away fromthe substratum are most important in the surface film. Advective transport dominates thesurface film.The bioflim compartment contains at least two phases: 1) a continuous liquidphase which fills a connected fraction of the bioflim volume and contains differentdissolved and suspended particulate materials. The suspended material consists of particleswhich can move in space independently of one another; 2) a series of solid compartmentseach composed of specific particulate materials, such as species of bacteria, extracellularand inorganic particles. The solids can not move freely, because the are attached to eachother. Movement of attached particles within one solid compartment causes displacementof neighbouring particles. Thus, each type of attached solid constitutes a different solidphase, which in addition may contain other compartments. Transport of a suspended6particle from the liquid phase to the solid phase within the bioflim has characteristics of areaction process, since educt and product do not belong to the same phase. Thus theinterfacial transfer processes must be distinguished from the transport processes within thebiofllm compartment.Bulk liquid compartment processes affect biofllm system behaviour primarily as aresult of the mixing or flow patterns resulting from the system’s geometry. Mass and heattransfer from the bulk liquid compartment to the biofllm compartment is dependent on theliquid dynamic regime: mass transfer in laminar flow will be much slower than in turbulentflow systems. The reactor or system geometry also influences mixing and, consequently,the mass transfer processes. Therefore, the reactor geometry and flow regime frequentlydetermine the progression of biofllm accumulation. The gas compartment provides foraeration and removal of gaseous metabolites, such as CO2. CH4, 112 in anaerobicbioflims.The biofllm system compartments interact with each other via transport andinterfacial transfer processes, which play a critical role in biofllm systems and generally,are rate-controlling (Characklis, W.G. & Marshall, K.C., 1990).1.1.2 The functions of bioflimsBiofllms represent a unique form of bacterial life. When adsorbed to surfaces,bacteria may change greatly in cellular physiological features (Gilbert, P., Evans, D.J. &Brown, M.R.W., 1993). It is the way in which bacteria adapt to the environment undercertain circumstances. The advantages of bacterial colonization on surfaces over discretecells, which can also be considered, to some extent, as the driving forces for bioflim7formation, are summarized (Fletcher, M., 1990; Flemming, Hans-Curt, 1993; Gilbert,P., Evans, D.J. & Brown, M.R.W., 1993) as follow:I) Nutrient enrichment Nutrients, especially surface-active substrate such ascations, fatty acids and macromolecules, are adsorbed to the surfaces and thusconcentrated and localized at the interface, facilitating utilization by attached bacteria. Onthe other hand, the bioflim functions as a filter in adsorption and uptake of nutrients fromthe flowing water phase and benefits the cell growth in the gel matrix.II) Enhancement of survival The bioflim structure provides some protection tobacteria so that they may become more likely than free-living cells to survive potentiallylethal conditions, such as short term pH-fluctuations, salt- and biocide concentrationshocks; shear forces and dehydration, etc..Ill) Development of microconsortia for improved growth, -symbiosis, - utilizationof less readily biodegradable substrate by specialized organisms, - creation of ecologicalniches.IV) They are not washed from the reactor by high flow rates of influent and havelong residence times.V) Pool and preservation of genetical information because of the long retentiontimes of the microorganisms and promoted genetic exchange brought about through closeproximity of other cells.VI) Modulation of the physicochemical environment of the cells through theestablishment and maintenance of pH and electro-potential gradients across the bioflim.1.1.2 The processes of bioflim formation and development8Although different views exist in the processes involved in biofilm formation(Characklis, W.G., 1990; Gilbert, P., Evans, D.J. & Brown, M.R.W., 1993; Bryers,J.D., 1993; Bryers, J.D. & Banks, M.K., 1990; Bryers, J.D. & Characklis, W.G.,1981), the biofllm formation and development can be divided into three stages: 1) bacterialadhesion to the surface, 2) the competitive success of colonizers and their subsequentgrowth, and 3) detachment and dispersal from bioflims.Bacterial adhesion to surfaces This stage includes three processes: 1.cell-particle transport to the substratum 2. reversible adhesion and 3. irreversible adhesionor attachment.Bacterial cells are transported to the substratum from the bulk liquid because offluid dynamic forces, gravity, Brownian motion, and bacterial tendency to adhere at thenutritious surfaces, etc. When the cells strike the substratum, they may adsorb to it, andthen desorb. This reversible process primarily involves long range interaction forcesbetween the cell and the substratum such as London - van der Waals force, double layerelectrostatic interactions, steric interactions, and possibly polymer bridging. Since this stepis mainly dependent on the thermodynamic properties of the related surfaces, it is alsoreferred as the non-specific adsorption.Some of the reversibly adsorbed cells may bond to the substratum firmly, thus theadsorption becomes irreversible. This step is frequently mediated by bacterial surfacepolymers, and is regarded as the result of specific interactions between cells andsubstratum. It involves short range force such as dipole-dipole (Keesom) interactions,dipole-induced dipole (Debye) interactions, ion-dipole interactions, hydrogen bonds,hydrophobic interactions, or polymeric bridging (Marshall, K.C., 1985).Here the colloid chemical theory - Derjaguin-Landau and Verwey Overbeek9(DLVO) theory, which describe the changes in the Gibbs energy of interaction as afunction of the distance between two particles, is utilized to explain the adsorptionprocesses (Characklis, W.G., 1990; Gilbert, P., Evans, D.J. & Brown, M.R.W., 1993).The DLVO theory has been successfully applied to microbial adsorption in describinglong-range forces while it remains a problem for short-range forces due to the changes inelectrochemical states resulting from physiological activities of viable microbial cells(Bryers, J.D. & Characklis, W.G., 1981; Rutter, P.R. & Vincent, B., 1984). For example,at low concentrations (<0. 1M) the affinity of Vibrio Alginolyticus for an hydroxyapatitesurface increased with ionic strength, in agreement with the D.L.V.O. theory. At higherconcentrations, bacterial affinity for the surface decreased with increasing concentration ofcations and was not related to ionic strength changes in the medium (Gordon, A.S. &Millero, F.J., 1984).Another basic theory for the quantitative description of the interactions of bacterialcells with substrata surfaces is the “wettability” theory, in which cell adsorption isconsidered as a function of the total interfacial free energy. This theory is often used forthe evaluation of surface characteristics concerning bacterial adsorption (Kozlyak, E.I. &Yakimov, M.M.; 1992; Characklis, W.G., 1990; Gilbert, P., Evans, D.J. & Brown,M.R.W., 1993).Competitive success and growth Following attachment to the substratum,bacteria will grow, modify the surface and propagate if conditions are suitable, resulting inthe development of microcolonies. At this stage, the production and accumulation ofextracellular polymers, usually polysaccharides, is often apparent. With time, growth andfurther attachment can lead to coalescing of microcolonies and complete coverage of thesurface by intracellular films of bacteria embedded in a highly hydrated polymeric matrix.10Thus the cells are immobilized with neighbouring cells in close proximity and with littleroom for growth. The dynamics and interactions with such biofllm communities are poorlyunderstood and almost untouched experimentally. Presumably, there are the followingsituations: 1) cometabolism and mutualism, exchange of metabolites, protons andhydrogen between functionally different bacteria, which may further stimulate growth ofmicroorganisms (Kent, C.A., 1988). 2) competition for nutrients, which may determinethe nature of the mature bioflim community. For a binary bacterial bioflim system, thefaster-growing bacteria will rapidly become dominant. However, the slower growingbacteria remain established within the biofilm and continue to increase in number over time(Banks, M.K. & Bryers, J.D., 1991). 3) production of antimetabolites which could allowa strain to resist adjacent colonization by newly attaching organisms.Detachment and dispersal Detachment from biofllms can be divided into threedistinctly different processes: erosion, the continuous removal of small particles from thesurface of bioflims, primarily caused by the shear stress created by water flow past thebiofilm; abrasion, caused by the collision and / or rubbing together of particles, some ofwhich are covered with biofllm; sloughing, periodic loss of large patches of biofilm.Detachment is often considered as the loss of biofllm resulted from the influences offoreign forces or the decay of the biofilm. To distinguish it from detachment, dispersal isused to describe the process of microorganisms spreading from the bioflim, which isbrought about through enzymatic or chemical cleavage control by microorganismsthemselves (Gilbert, P., Evans, D.J. & Brown, M.R.W., 1993). Dispersal is a selfadaption mechanism of bacteria in order to survive and colonize new niches. Some studiessuggested that changes occurred in the adhesive properties of the cell surfaces, associatedwith the cell division process, and were able to bias the reversible adhesion in favour of11dispersal (Eighmy, T.T, Maratea, D., & Bishop, P.L., 1983).The rate of detachment is influenced by the foreign forces to which bioflim isexposed. The surface of the substratum also provides physical conditions which affectdetachment although the impacts may be more subtle than for the hydrodynamic effects ofshear stress and abrasion. Similarly, the physiological factors of bacteria, such as specificgrowth rate, extracellular polymer production may play an important role in determine thedetachment rate.1.1.3 The factors affecting the bioflim processesThe factors governing bioflim formation include (Fletcher, M., 1990; Wilderer, P.A. &Characklis, W.G., 1989):• Inherent genetic characteristics, which ultimately determines the bacterial surfacecharacters• Genetic expression mechanisms, which control the adhesiveness of bacteria inresponse to different environments• Physiological factors such as growth rate, cell concentration, age• Nutritional factors such as carbon, nitrogen, minerals• Physicochemical factors such as pH, temperature, ionic strength, cations• Surface characteristics of substrata such as surface chemical properties, roughness• Physical conditions such as flow pattern, mixing, reactor geometryConsidering their importance to practical biological operations, only some of theabove factors were selected for discussion. Other factors may be covered in discussions of12other topics if related.Surface characteristicsThe DLVO theory and wettability theory provide guidelines for the analysis of theeffects of surface characteristics of both substrata and bacteria.[Substratum surfaces] The properties, namely surface characteristics, of supportmaterials affect mainly the formation of the first layer of bioflim. The surface charge andthermodynamic parameters used to measure substratum differences include interfacial orsurface free energy, critical surface tension, water wettability, water contact angle, etc. Asfar as surface tension is concerned, in the range of 20 -30 dyne/cm, the adhesion ofmicroorganisms is less favourable of. Most engineering materials and coatings show a highcapacity for adsorbing the first layers of biofilm.Higher surface roughness favours the anchorage of microorganisms due to theincrease in contact area. However this effect is limited to the first layers of bioflim andtherefore to the induction of the overall process. Thus, the use of small size porousgranulated carriers and porous fibre carriers was recommended for higher capacity ofbacterial adsorption. The minimum pore size of five times larger than the major dimensionof the cells reproducing by division was noted (Messing, R.A., 1988). The toxicity ofsome metallic ions depend on the dominant microbial species and on the environmentalconditions. It is thought that the toxic effect of the metallic ions ceases after several layersof biofilm are formed. In this case, this effect may be responsible for retarding the overallprocess without eliminating it completely.[Bacterial surfaces] The effect of bacterial surface characteristics are significant not only13at the initial stage of attachment, but also during community development. The cellsurface characteristics are greatly variable, depending on genetic determinants and thecellular physiological and morphological status as a result of phenotypical responses tochanges in environmental conditions. However, similarity in cellular surface characters stillexists among different types of bacteria and are beneficial for general considerations ofbacterial adsorption. The bacterial surfaces and fibres of polysaccharides are usuallyconsidered to be negatively charged. Therefore, use of positively charged surfaces wouldfacilitate the adhesion of bacteria. Many studies suggested that slightly more-hydrophobiccells are preferable for adsorption on surfaces, especially on hydrophobic surfaces(Kozlyak, E.I. & Yakimov, M.M., 1992; Gilbert, P., Evans, D.J. & Brown, M.R.W.,1993).Nutrient concentrationThe effect of growth limitation on bacterial adhesion was investigated underconditions of limitation of various nutrients, mainly C- and N- sources and oxygen(Applegate, D.H. & Bryers, J.D., 1990; Kozlyalc, E.I. & Yakimov, M.M., 1992;Kjelleberg, S., 1984). It was found that growth under C- and N-limitation led to drasticchanges in cell surface properties and adhesion patterns. In some cases, C-limitationresulted in the maximum adsorption of bacteria whereas growth limitation due to N-source worsened the adsorption. Possibly, under such conditions bacteria switchedmetabolism to synthesize reserve polymers, adhesive polysaccharides for C-limitation(Talcil, S., 1977) and non-adhesives for N-limitation (Kozlyalc, E.I. & Yakimov, M.M.,1992). In some cases the incubation of cells in a starvation medium or tap waterremarkably increased the cell adhesiveness.As the microorganisms become attached their growth will depend on the diffusion14of the nutrient through the film. The limit for this difihision depends on the thickness andon the structure of the film. As a rule, when a bioflim thickness is more than 50 jim,diffusion limitation occurs (Peyton, B.M. & Characklis, W.G., 1993). The low level ofnutrients in the deeper zones of these films may result in a decrease in the production ofexcreted polysaccharides thus rendering the films more vulnerable to shear forces. Wandaet al. found that a more complex substrate causes a better bioflim development andpopulations with a high tendency to adhesion showed a high total exopolysaccharidecontent (Wanda, U., Wollersheim, R., Diekmann, H. & Buchholz, K., 1990). Thestudy of biofilm activity in a three phase fluidized bed showed that the substrate flux andthe detachment rate controlled the amount of biofilm colonization. However, only the fluxaffected the biofilm activity, observed yield, and 02 consumption. The higher the flux, theless inactive is the biomass, because substrate concentration is higher in the biofilm andwhich allows the cells to maintain a higher specific growth rate. The amount of biofilmcolonization also increased when the biofilm became more active because of having alarger substrate flux (Rittmann, B.E., Trinet, F., Amar, D., & Chang, H.T., 1992).Cellular physiological statusAn understanding of the effect of the growth states of suspended cells on initialformation is important for the practical application of biofilms. In a number ofexperiments, it was observed that bacterial cultures at the exponential phase of growthhad the greatest facility for adhesion, followed by stationery and then death-phase cultures(Kozlyalc, E.I. & Yalcimov, M.M., 1992; Fletcher, M., 1977). However, theadhesiveness of certain bacteria which remained unchanged at all their growth stages werealso reported. On the other hand, it was also found that the detachment rate of bacteria,such as marine Pseudomonas aeruginosa, is directly related to biofilm growth rate and15that factors which limit growth rate will also limit detachment rate (Peyton, B.M. &Characklis, W.G., 1993). That the adhesiveness and surface hydrophobicity ofStaphococcus epidermidis and E. coil decreased in early- and mid- exponential phase(Gilbert, P., Evans, D.J., Evans, E., Duguid, I.G., & Brown, M.R.W., 1991)provided another example of the effect of physiological status on bacterial attachment. Inthis case, cell surface charge became more electro-negative for E. coil but electro-neutralfor Staphococcuc epidermidis as the cells proceeded to divide.Fluid velocityLow velocities tend to favour initial colonization of surfaces due to thecharacteristics of the laminar layer that facilitates the anchorage of microorganisms. Butthe laminar layer constitutes an obstacle to the diffusion of the nutrients and oxygenindispensable to the metabolism of the attached cells. Higher velocities are morefavourable to the renewal of nutrients near the surfaces, allowing a faster growth of themicroorganisms. However, high velocities produce higher shear stress which would inducethe removal of biofilms - erosive effect. The results from experiments in an outdoor openchannel, simulating natural river conditions indicate that biofilm biomass accumulation wassubstantially reduced as flow shear stress increased and that the maximum accumulationoccurred under very low flow conditions (Lau, Y.L. & Liu, D., 1993). In another case,the rate of cell removal by fluid shear for a species was found to be a function of bioflimcell number only if the species concentration was uniform with depth; in essence, only theupper layers of the biofilm were sheared off (Banks, M.K. & Bryers, J.D., 1991). Nosignificant influence of shear stress on detachment rate was observed in the experimentswith Pseudomonas aeruginosa (Peyton, B.M. & Characklis, W.G., 1993). In general,higher velocities tend to produce thinner films than lower velocities but might increase the16microbial activity in the film. Higher velocities lead to compaction of the biofilm, or higherbiofllm densities for higher velocities.The structure of a bioflim ( also dependent on the fluid velocity) has a relevanteffect on the growth and reproduction of the attached microorganisms. The structure ofbiofilms is not uniform along the thickness of the biofilms resulting in a more compactlayer near the deposition surface and a somewhat looser one near the solid-liquid interface.A more compact structure may be a limiting factor for difihsion of the nutrientsthroughout the biofilms. Thus, for moderate velocities, cell growth may increase in thepresence of higher fluid velocities, but for much higher velocities the “active” layerbecomes limited to the superficial layer of the bioflim (Kozlyak, E.I. & Yalcimov, M.M.,1992).pH of the fluidThe influence of fluid pH is associated with the growth rate of the microorganismsand the adhesion forces at the surfaces. a). Greater amounts of deposits are formed at thepH coinciding with the best values for growth and reproduction of the microorganismspresent in the film. The pH conditions in the solid- liquid interface may be altered by thecellular metabolism which may produce acids or alkalis. b). pH can affect the distributionof electrical surface charges of materials present, and thus, their zeta potentials. Theadhesion between these materials is increased when the corresponding zeta potentials haveopposite signs. Since most usually, the majority of bacteria have negative electrical surfacecharges, it can be said that the coating of solid surfaces with materials that increase theelectronegativity, would reduce the possibilities of adhesion of the great majority ofbacteria.17TemperatureThe effect of temperature is usually concerned with the influence of fluidtemperature on the growth of the microorganisms and for some cases, the production ofexopolymers by the cells. Therefore, the optimum temperature for the bacteria growth ismostly taken as that to promote adhesion of the cells to surfaces.Presence of inorganic suspended particlesMicrobes have a tendency to cover suspended inorganic particles, formingaggregates. This would enlarge the induction period of biofllm formation. On the otherhand, higher quantities of bioflim are obtained when the asymptotic values of the bacteriaattachment is reached. This may due to the new type of bioflim structure or to thestimulation of attached cell growth and metabolism in the presence of inorganic particles.The growth and reproduction of cells in the film is increased due to the presence of theinorganic particles. This stimulation is related to the structure formed in the bioflim thattends to be less compact and therefore more favourable to the diffusion of nutrients. It canalso constitute a positive factor for microbial growth, inorganic particles may function as asource of nutrients when entrapped in the film. Inorganic particles, such as kaolin particlestend to become not only colonized by microbe but also coated with organic moleculeswhen in suspension.1.2 Bioflims for anaerobic wastewater treatmentWastewater treatment with bioflim reactors is one of the most large-scale andoldest applications of biofllms in industry. In this area, anaerobic biofllm reactors have18become more and more important in the last decade. This reflects the recognition of theiradvantages over the traditional biological treatment processes and the advancement inresearch and development of biofilm technology.1.2.1 Anaerobic digestion and related bioflim reactor technologiesThe anaerobic digestion process of organic wastes is divided into three stages orphases, based on the metabolic reactions characterized by different groups of bacteria: 1).acidogenesis - hydrolysis of macromelecular compound so that they can be transportedinto the cell through cytoplasmic membrane, and conversion of these initial degradationproducts into intermediates, namely small molecular weight organic acids, carbon dioxideand hydrogen 2) acetogenesis - further degradation of the intermediates into acetate,carbon dioxide and hydrogen, and 3) methanogenesis - production of methane bydecarboxylation of acetate and reduction of carbon dioxide (Gujer, W. & Zehnder,A.J.B., 1983; Marty, B., 1986; Boone, D.R., 1985; Price, E.C., 1985; Large, P.J.,1983). This model is widely accepted because it better reflects the interactions of differentbacterial species in anaerobic digestion (Thiele, J.H., 1991). However, in practicalapplications, the traditional two-phase model, which combines the phase two and three inthe three-phase model as a single methanogenic phase is more often used. This is becausethe two-phase model emphasizes the important distinction between the microorganisms ofeach phase, which are vital for design and control of the digestion process in terms ofreaction kinetics. For instance, the acidogenic bacteria generally grow faster and are moreresistant to inhibition, compared with the slow growing and fastidious methanogens. Thereactor loading for acidogenesis can be 4 - 6 times higher than that for methanogenesis(Henze, M. & Harremoes, P., 1983).19The basic types of anaerobic bioflim reactors applied to wastewater treatmentinclude: fluidized bed, expanded bed, upflow anaerobic sludge blanket (UASB), fixedfilm/filter, rotating biological contactor, and hybrids. Their configurations, advantages andperformances can be found in the excellent reviews (Henze, M. & Harremoes, P., 1983;Bhamidimarri, S.M.R., 1990).1.2.2 Characteristics of biofilms in anaerobic bioflim reactorsDirectly measured data on the structure and properties of biofllms for anaerobicwastewater treatment are still quite limited. The anaerobic bacteria may follow the generalpatterns of bioflim formation and consequently exhibit similar bioflim characteristicsalthough diversity exists.In a study on the activity and the structural characteristics of methanogenicbioflims on needle punched polyester supports in a downflow-stationary fixed-film reactor(Harvey, M., Forsberg, C.W., Beveridge, T.J., Pos, 3., & Ogilvie, J.R., 1984), fewbacteria were found to be tightly adherent to the support surfaces. However, there was amorphologically complex, dense population of bacteria trapped within the polyestermatrix. Frequently large microcolonies of a uniform morphological type of bacteria wereobserved. These were particularly evident for methanosarcina-like bacteria which grewforming large aggregates of unseparated cells. Leafy deposits of electron-dense, calcium-and phosphorus-enriched material coated the polyester matrix and some cells. As thebiofilm matured there was a more extensive material deposition which completelyentrapped cells. The trapped cells appeared to autolyse, and many were partially degraded.The results from energy dispersive X-ray (EDX) analysis of the dense precipitate and the20cells indicates that calcium and phosphorus concentrations in the extracellular matrix were92 and 28 times higher respectively than in the cells. Calcium carbonate and calciumphosphate are probably the major components of the precipitate. It was postulated thatfurther impregnation of the matrix with minerals and cell death may eventually have adeleterious effect on the methanogenic activity of the biofilm. The formation of biofilm isthought to begin by adhesion of single cells, with the gradual formation of a monolayer ofcells. This leads to colonization by other organisms and the subsequent development of athick film. The driving force of colonization is believed to be the adsorption of nutrient tothe surfaces. As the population density increases on the support, there is an enhancedopportunity for cross-feeding, cometabolism, interspecies hydrogen transfer, andinterspecies proton transfer, which may further stimulate growth of microcolonies.This postulation coincides with what was suggested by the results from electronicmicroscopic observation of the ultrastructure of bacterial granules in an UASB and filterreactor (Macleod, F.A., Guiot, S.R., & Costerton, J.W., 1990). SEM (scanningelectronic microscopy) and TEM (transmission electron microscopy) studies have revealedthat the granular aggregates were three-layered structures. Based on these observations, astructure model to explain the granule development was proposed. The aggregate consistsof mostly Mathanothrix in the centre core of the granule, acetogens and hydrogenconsuming bacteria in the middle layer, and acidogens and hydrogen consuming bacteria inthe exterior layer. Methanothrix might function as nucleation centres that initiate thedevelopment of the granule. The loose mat of Methanothrix filament provided aframework for colonization of other organisms. The first colonizing bacteria such as 112-producing acetogens provided the methane producing bacteria with the required substrate,acetate. Because the high concentrations ofH2 generated by theH2-producing acetogensinhibit degradation of propionate and butyrate, the syntropic association with H2-using21bacteria would be required. The existence of this group of bacteria in the granule wasconfirmed. The metabolism of fermentative bacteria in the exterior layers of the granulewould produce the substrate for underlying acetogens. The presence of}12-using bacteriacould consume free hydrogen before it penetrated into the second layer.The results from the investigation of irreversible attachment to glass slides andbioflim development of anaerobic bacteria showed that the slow rate of development ofthe methanogenic consortium attached to a surface ( probably due to long doubling timesof the methanogenic bacteria) is a more significant, ultimate limiting factor in the start-upof a methane producing bioflim reactor than the rate of bacteria attachment, as externalmass transfer resistance of substrate may also limit the developing bioflim followingbacterial attachment. On the other hand, proper preparation of support surfaces isimportant for bacterial attachment (Robins, J.P. & Switzenbaum, M.S., 1990).To study the effect of solid supports on adhesion of methanogenic and acidogenicbacteria, many experiments have been conducted. For example, Yu and Pinder (1992)examined quantitatively the selectivity of attachment of acetogens and methanogens oninert support surfaces. They found that the surface preference of the bacteria species used(degrading acetate, propionate and butyrate) decreased in the sequence of wood, ceramic,PVC and stainless steel, mainly due to the different hydrophilicity and wettability of thesematerials. Kuroda et al. submerged test specimens into bacteria slurries cultured indifferent media and found that bacteria adhered to the moderately rough surfaces withpores measuring a few tenths of a micron in diameter better than to the polished surfacesand very rough surfaces, and preferably adhered to the solid supports made ofcarbohydrate material. The accumulation rates of biomass on the solid supportssubmerged in the mixed slurry of acid-producing bacteria and methane-producing bacteriaare higher than those on the solid supports submerged in a slurry of methane-producing22bacteria (Kuroda, M., Yuzawa, M., Sakakibara, Y., & Okamura, M., 1988).Therefore, the formation of the bioflim depends not only on the characteristics of thebacteria and fluid regimes but also on the characteristics of the solid supports. In anothersimilar experiment with different types of support materials in down flow stationary fixedfilm anaerobic digesters, wood chips were found to be a more effective support materialthan charcoal and ceramic Rasching rings. The acid conversion efficiency improved withhigher retention times, but the productivity declined (Scharer, J.M., Bhadra, A. &Moo-Young, M., 1988).Also, film development, indicated by the rate of converting acetate to methane andC02, was 3 times faster on fired clay than on either PVC or etched glass. SEMphotographs showed that the bacterial film on clay was thick and uniform, while the filmattached to PVC plastic was thin and still uniform. Attachment to etched glass was Spotty.The characteristics of clay which made it a superior support appeared to be its rough,porous surface which offered attachment sites to the microorganisms and the presence ofminerals in the clay, particularly Fe which is known to stimulate methanogenesis andgrowth (Murray, W.D. & van den Berg, L., 1981).A study on the population dynamics of attached bioflims in an anaerobic fluidizedbed pilot plant showed that the biofilm biomass contributed to more than half the amountof total biomass in the reactor while the suspended sludge was susceptible to hydraulicwashing-out. Filamentous networks and thrix-like anaerobic bacteria grew initially in thedeep holes or crevices of the medium. After this initial bioflim network formation, certaintypes of short rods and small cocci were embedded in the biofilm. High organic loadingand high WA (volatile fatty acids) concentration provided rapid growth of discrete rodswhich were washed out of the fluidized bed (Chen, S.S., Huang, S.Y., Lay, J.J., Tsai,P.S., & Cho, L.T., 1992).23In summary, in addition to possession of some general bioflim characteristics suchas gel matrix structure due to highly hydrated polysaccharides, diffiisional limitation, celladhesion subjected to changes in support properties, etc., the anaerobic bioflim has thefollowing characters: 1). very slow growing due to the nature of the anaerobes 2). thelayered structure as the result of the mixed-culture 3). cavities in the bioflim broughtabout by the presence of gases 4).the interactions between species in the bioflim tend tobe more commensal than inhibitory. 5). the bioflim contains a large amount of mineraldeposit, especially calcium salts.1.2.3 Modelling of anaerobic digestion in bioflim reactorsDue to the complexity of anaerobic digestion by bioflims and the difficulties inexperiments with anaerobic microbes, modelling the bioflim processes has been of greatinterest in research and development. Many models for the quantitative description ofanaerobic biofllm processes have been established on the basis of the kinetics of bacterialgrowth and biochemical reactions involved in anaerobic digestion, principles of colloidalparticle thermodynamics, principles of mass transfer processes and fluid hydrodynamics,and the characteristics of anaerobic biofilm structures (Bouwer, E.J., 1987; Kitsos,H.M., Roberts, R.S., Jones, W.J., & Tornabene, T.G., 1992; Yu, J. & Pinder, K.L.,1993; Furumai, H. & Rittmann, B.E. ,1992), and quite often were made by modifyingthose for aerobic bacteria (Wanner, 0. & Gujer, W., 1986; Chang, H.T. & Rittmann,B.E., et al., 1991; Williamson, K. & McCarty, P.L., 1976; Rittmann, B.E. &McCarty, P.L., 1978; W.G. Characklis & K.C. Marshall , 1990). Since the substrateremoval rate is the factor of greatest concern in the evaluation of the performance of an24anaerobic bioflim digester, early work focused mostly on kinetic models regardingsubstrate consumption, biofilm growth and product production.Most recent kinetic models can be classified in four levels: I). including a simplemass balance equation on the substrate and the logistic equation or the Monod equation orits modified form such as one incorporating the product inhibition factor. In this case, it isoften assumed that all the biomass exists in the form of biofilm and is viable. II).incorporating the Fick’s equation for diffusional transport of substrate, into level 1. III).considering the interaction of the bacteria in two different phases or bioflim layers (Fang,M., Howell, J.A. & Canovas-Diaz, M., 1989). IV) incorporating the loss of bioflim as afunction of shear force and/or the decay ofbiomass in the models. The models above level2 become very complicated and some of the parameters are difficult to measure. Inpractice, some simplified models are more useful for analysis of substrate consumption andbiofilm growth under specific conditions.1.3 Calcium and bioflimsAs mentioned above, calcium is one of the most often found inorganic elements innatural biofilms. Whether this is due to its abundance and wide distribution or because ofits roles in the biofilm system, the effect of calcium on biofilm formation and bioflimactivity is an interesting unknown with respect to the control of bioflim processes.1.3.1 The bacterial roles of calcium25The biological roles of calcium have been clearly defined as: 1) structural -Structure of soft tissues ( cell adhesion, membrane permeability), 2) electrical - electricalactivity across some membranes, 3) cofactor for extracellular enzymes and proteins, 4)intracellular regulator (Campbell, A.K., 1983). However, these theories are establishedmainly on the basis of the studies on human and animal cells. For bacteria, calciumsfunctions are far less understood, although calcium is always used in bacterial culturalmedia (Silver, S., 1977).Norris et al. summarized a large number of diverse processes in bacteria in whichcalcium is involved, including chemotaxis, sporulation, phosphorylation, heat shock, theinitiation of DNA replication, septation, nucleoid structure, nuclease activity andrecombination, the stability of the envelope, and phospholipid synthesis and configuration.They pointed out that since such varied processes have a common factor, calcium,suggesting that there are some major underlying principles of calcium metabolism whichhave not yet been discovered (Norris, V., Chen, M., Goldberg, M., Voskuil, 3.,McGurk, G., & Holland, I.B., 1991). Another author mentioned that Ca2 might alsofunction as an intracellular messenger (secondary messenger) in a microbial system, and beinvolved in processes such as ionic inducing bacterial mating (J.L. Reissig, 1977). Sprott,GD. examined the structure and function of some methanogens’ cell surfaces. He foundthat the sheaths of the methanogens contain predominant amount of Ca2+ and that Ca2+played a role in countering the toxic effects of high concentration of ammonia (Sprott,G.D., 1986). The Ca2+ concentrations for maximum growth of methanogenic bacteria inbatch culture are 1 mM for Mc. voltae in CO2LFI substrate, 13.6 j.iM for Ms. thermophilein methanol, 0.25 mM for Mtx. concilii in acetate, and 2.5 lLM for Mtt concilii in acetate(Sprott, G.D., 1989).Calcium is normally transported out of bacterial cells and the intracellular level of26calcium is very low. For example, the intracellular level of free calcium in E. coil is verytightly regulated to i0 M (Rosen, B.P., 1984). The amount of Ca2 required to give100 g of dry biomass when Ca2 is growth limiting is about 0.1 g, much lower than thatof K and Mg2 (Beveridge, T.J., 1989). According to Campbell (1983), calciumconcentration in the cytoplasm is usually 1-10 mMole/l cell water. Takashima and Speece(1990) reported that calcium is required for stability of methyitransferase in methanogensand bacterial aggregation (Takashima, M. & Speece, R.E., 1990); Calcium content inmethanogens belonging to order 1 is 85 -550 Ig/g dry mass, and order 2 & 3 1000-4500lig/g dry mass. The optimum calcium concentration for Mc. voitae (H2/C0)is 40 mg/iand Ms. thermophila (methanol) >0.45 mg/I.It is believed that all microbial calcium functions are at the cellular membrane orexternal to the membrane. There is no required intracellular role for calcium in bacterialcells (Silver, S., 1977).In biological systems, cations Na, K, Mg and Ca are distributed selectively, with Kand Mg concentrated inside the cell and Ca and Na outside the cell. The systems aremembrane bound and transport Ca2+ against a four orders of magnitude concentrationgradient, out of the cytoplasm into extraceliular fluids or specialized intracellularcompartments. The mechanisms involved are based on the operation ofCa2+activ tedATPase utilizing the chemical potential of ATP, or countertransport systems utilizing thepotential of other primary electrochemical gradients for transport of Ca2+ against its owngradient. Another and complementary aspect of Ca2+ regulation is the controlled flux ofCa2+ back into the cytoplasm, when Ca2+dependent systems in the cytoplasm must beactivated. These Ca2+ fluxes occur down the electrochemical gradient and, in this respect,may be considered passive’. Therefore, they are regulated basically by exchanges inmembrane permeability. A large body of evidence indicates that these passive fluxes occur27through specific channels, whose gating is voltage dependent, as well as regulated byhormones and drugs (“Calcium blockers” ) (Rosen, B.P., 1984). Relatively little is knownabout bacterial transport of Ca2+. Some mechanisms proposed for calcium cationmovement across prokaryotic cytoplasmic membranes include secondary cation/protonantiport activity and other antiporters: calcium/proton, calcium phosphate/proton (in E.Coli), and sodium/calcium (in Halobacterium halobium) (Sprott, G.D., 1989).1.3.2 Previous studies on the effects of calcium in bacterial bioflimsThe studies on the effect of calcium on granule development in UASB reactors(Hickey, R.F., Wu, W.-M., Veiga, M.C., & Jones, R., 1991; Huishoff Pol, L.W., deZeeuw, W.J., Veizeboer, C.T.M., & Lettinga, G., 1982) showed that lowconcentrations (80 -200 mg/i) of calcium appeared to be beneficial for the development ofgranules (Lettinga et al., 1980; Hulshoff et al., 1983). However, lab-scale experiments toform granules from a digested sludge demonstrated that a calcium level of 450 mg/Iincreased sludge wash-out (HulshoffPol et al., 1983). High calcium concentrations (800 -1000 mg/i) induced a decline in specific activity of granular sludge (Lettinga et al., 1985;Thiele et al., 1990).For expanded/fluidized bed reactors, wastes containing high level of calcium areparticularly difficult to treat. Wastes with 2.5 g/l calcium were able to be effectivelytreated on a laboratory scale, however, after 150 days calcite precipitates were observedto be about 30% of the total weight of sand/biomass particles (Jordening, et al., 1988).Greater than 90% of the calcium in a waste containing 0.9 - 3.0 g/l calcium was reportedretained in a 10-litre laboratory-scale reactor. The accumulation of calcium resulted in28increased particle density, loss of fluidization, clogging and 40% dead space (Vogel andWinter, 1988) There is serious doubt whether the expanded/fluidized bed systems canfunction effectively over a long time frame with high-calcium wastes.A distinct improvement in sludge settleability and specific activity was observedafter replacing Na2CO3 as a neutralizing agent by Ca(OH)2 in the treatment ofpotato-processing wastes in a 6 m3 pilot plant (Versprille, A.I., 1978). A similar resultwas obtained when Ca(OH)2was utilized for neutralization during the start-up period ofUASB reactors (Salkinoja-Salonen, M.S., Nyns, E.-J., Sutton, P.M., van den Berg,L., & Wheatley, A.D., 1982). These results may imply the positive effect of calcium.Guiot, S.R., Gorur, S.S. and Kennedy, K.J.(1988) investigated the effects of thepresence of calcium ions ( 80 mg/I ) on microbial aggregation during upfiow anaerobicsludge bedfllter reactor start-up. The results show that calcium cations had no significanteffect on granulation, at least as an inducer of granulation at low substrate consumptionrates. They thought that certain specific conditions were required before calcium ionscould accelerate the granulation process, because two factors might impair the Ca2+action. Firstly, in bicarbonate-buffered systems, calcium partly precipitates. Secondly,sodium cations abundantly present in the medium might compete for binding sites withCa2without bridging occurring.Turakhia and Characklis investigated the effect of calcium on the biofllm activity ofPseudomonas aeruginosa (Turakhia, M.H. & Characklis, W.G., 1988). The resultsindicated that specific activity in the biofllm was the same as that measured in a chemostatand was not influenced by changing calcium concentrations. However, increasing calciumconcentration increased the cohesiveness of the bioflim.Applegate and Bryers studied bacterial bioflim (pure culture of the obligate aerobePseudomonas putida ATCC 11172) removal processes due to shear and castrophic29sloughing in a turbulent flow system under conditions of carbon versus oxygen substratelimitations and varying aqueous phase free calcium concentrations. The results showedthat increasing free aqueous phase calcium concentration increases the amount ofbioflim-bound calcium. The rate of calcium binding in02-limited bioflims increases withincreasing free calcium concentrations over the entire range studied ( 5 - 13 mgll) whilethe rates of calcium binding in C-limited biofilms are independent of free calciumconcentrations above 8.0 mg/l. 02-limited biofilms, with higher extracellular polymercontent and bound calcium, exhibit a much lower shear removal rates than the C-limitedbiofilms. However, they always experience catastrophic sloughing events. They proposedthat reduced shear removal and the susceptibility to sloughing in the02-limited biofilmwere attributed to their denser, more rigid crystalline structure brought about by excessivepolymer production and concomitant binding of calcium (Applegate, D.H. & Bryers,J.D., 1990).Experiments with a marine bacteria showed that omission of Ca2+ and Mg2+from the artificial seawater prevented growth, polymer production and sorption tosurfaces by the organism (Marshall, K.C., Stout, R., & Mitchell, R., 1971). In anothercase, addition of Ca2+ (as CaC12 ) in concentrations exceeding 128 pm producedsignificant increase in the adhesion of the test strains to plastic (Dunne, W.M., Jr., &Burd, E.M., 1992).The importance of Ca2+ (and M82jin adhesion was also clearly demonstrated inan electron microscope study of the marine Pseudomona species. The cells formed the‘primary’ material; then after adhesion, the ‘secondary’ polymer. When transferred tocation-deficient medium, the secondary polysaccharide was greatly disrupted in a veryshort time, indicating a role for the ions in maintenance of the adhesive structure. Underconditions of divalent cation deficiency, the polysaccharide-containing polymers30associated with irreversible adsorption were not detected. Some marine bacteria remainattached when washed with sea water but can be removed by treatment with tap water.This may indicate a requirement for critical concentrations of certain ions if adsorption isto be maintained and could also involve highly charged polysaccharides (Sutherland,I.W., 1983).Many researchers believe that Ca2+ may participate in the formation of the cationbridge which linked two polysaccharide fibres of two cells (Costerton, J.W., Geesey,G.G., & Cheng, K,J., 1978). On the other hand, some researchers think that it is unlikelythat calcium is involved in direct bridging to a negatively charged substratum. Instead,calcium maintains the tertiary structure of extracellular polymer substances so that theinteractions between the adjacent sugars on different chains are promoted (Turakhia,M.H., Cookey, K.E., & Characklis, W.G., 1983).Calcium cations are also likely to be involved in the modification of the substratumsurface and facilitate bacterial adhesion in the initial formation of biofilms because of itsadsorption to surfaces (Hermesse, M.P., Dereppe, C., Bartholome, Y., & Rouxhet,P.G., 1988). Furthermore, the presence of calcium may alter the ionic strength of the bulkliquid phase and subsequently affect the bioflim formation process (Kahane, I., Gat, 0.,Banal, M., Bredt, W., & Razin, S., 1979).To sum up, the presence of Ca2+ in most cases had a positive effect on biofllmprocesses due to the roles of calcium in bacterial cells and in the biofilm formationmechanisms. The data on the measurement of such effects are very limited. Further studiesin this area would be useful for analysis and control of bioflim processes in wastewatertreatment, biofouling and the biotechnology applications of immobilized cells.31CHAPTER 2EXPERIMENTAL2.1 Experimental objectivesIn this study, the effects of calcium on the biofilms in anaerobic fixed-bed reactorsfor the acidogenesis of lactose were investigated. While the study mainly focuses on therelationship between the bioflim accumulation and the calcium concentration in the culturemedium, the influence of calcium on the bioflim activity, density and immobilized calciumcontent are also examined.Acidogenesis of lactose under anaerobic conditions is selected for this studybecause lactose is the major component of cheese whey, a dairy plant waste, which isgenerated in large quantities worldwide. Anaerobic biofllm reactor technology has beenused to treat cheese whey at both laboratory and industrial scales. In the methanation oflactose, acidogens usually grow much faster than methanogens, and the accumulation ofexcess inactive biomass in acidogenic biofilm reactors was thought to be responsible forthe reactor efficiency decline. Anaerobic digestion of lactose by acidogens has been wellstudied in the chemostat (Murray, W.D. & van den Berg, L., 1981) and bioflim reactors(Barthakur, A., Bora, M., & Singh, H.D., 1991).In order to achieve the above objectives, the bioflims were grown in a reactorsystem, where only the calcium concentration in the culture medium was varied in theexperiments while the rest of the experimental parameters were kept constant. To observe32the influence of changes in the calcium concentration on established biofllm development,some of the biofilms formed in the culture medium with one calcium concentration weremoved into the culture medium with a different calcium level for their fhrtherdevelopment.2.2 Experimental apparatus and conditions2.2.1 Experimental set-upThe experimental set-up used in this study is illustrated in figure 1. It wasoriginally designed by Yu and Pinder (1991). The system consists of the followingelements:1) two parallel continuous flow reactors for the biofllm growth (biofllm reactors).Each reactor, with an effective volume of 1.5 litres, was made of a half Plexiglass tube,which provides the reactor with a curved bottom so that deposition of bacterial sludge inthe reactor can be minimized. The reactor was equipped with a circulation pump (ColeParmer Masterfiex pump) for mixing and control of fluid flow velocity, a pH controller(Cole Parmer Series 7142 pHlPump System) with 1 N caustic solution (50% NaOH and50% KOH), a water bath thermostat (COLORA) for temperature control through anexternal heat exchanger, and a nitrogen gas disperser for displacing oxygen in the reactor.2) biofllm supports and sampling ports. 1.587 millimetre thick PVC slides(4. 5x 1.5 cm) were utilized as the biofilm supports in the reactors. They were held at oneend in rubber stoppers and immersed into the culture medium through the sampling portsarrayed in the top of the reactor.33GasCollectorpHControllerCa2+CoolantBase/AcidTemp.ControllerEffluentPVCslideFigure1IllustrationoftheExperimentalSet-up3) feed supply system. The culture medium for both of the reactors was stored inthe feed tank and kept at 1 - 4 °C by the coolant circulating from the cooling bath(NESLAB) through an immersed coil. Each loading of the feed in the tank was limited tothat needed to supply the reactors for three to four days. Nitrogen was introduced into thetank to purge oxygen from the medium. The flow rate of the feeds was controlled withtwo pumps (Cole Parmer Masterfiex). Two sampling ports were installed before the beaktubes, which prevent the feed in the storage tank being contaminated. The sampling portswere also used for monitoring the feed flow rate by measuring the fluid volume over aperiod of time.4) calcium addition. Additional calcium solution was added into the feed for thespecified reactor by using a precise dosing pump (miniPump, Milton Roy).2.2.2 Experimental conditionsIn each of the bioflim growth experiments, the reactor operating conditions wereset to optimize the bacterial growth in the reactors and consequently promote theattachment of cells to the support surfaces.Generally the optimal pH for acidogenesis is 4 - 8, the optimal temperature for themesophilic bacterial growth is 32 -39 °C. According to Kisaalita, Pinder and Lo (1988), alower pH value (4.5) in the range of 4 - 6.5 for acidogenesis of lactose would result in aproduct distribution which might favour the methanogenesis process. From the stimulationtests, Yu and Pinder (1991) concluded that to ensure a completely mixing pattern in thereactors and to minimize the external mass transfer resistance on substrate utilization inthe biofllms, the feed flow rate should be below 1.7 I/hr while the recycle rate should be35kept above 14 IJhr. And in this case, the substrate concentration should be about 10 g/l inorder to eliminate growth limitation due to the substrate. Therefore, the reactor operationconditions were set as shown in table 1.The composition of the basic culture medium is shown in table 2. This formula wasused by Kisaalita, Pinder and Lo (1988) and Yu and Pinder (1991) in the previous studieson acidogenesis of lactose. It was believed that such a formula should satisfS’ therequirements of the balanced growth of acidogenic bacteria for the substrate and nutrients.The lactose concentration was varied depending on whether the medium was used for themicrobial inoculum preparation, bioflim growth or the assay of the bioflim activity. Theconcentrations of the organic salts were then adjusted proportionally according to theformula.Table 1 The reactor operating conditionspH 4.5±0.2temperature 35 ± 1 °Cfeed flow rate 150 mllhror dilution rate 0.15 hr 1recycle rate 230 mllmin.substrate concentration in the feed 5 - 10 g/lBased on the findings from the previous studies (see 1.3.2), the calciumconcentrations in the feed for the first run of the experiment were set as follow: high level36-- 100 mg/i; control -- no added calcium. In the following experiments the selection ofcalcium concentrations was based on the results of the earlier experiments. They areshown in table 3. It should be noted that these calcium concentrations are much lowerthan the calcium level reported to inhibit suspended anaerobic bacteria in batch orchemostat culture (Kugelman, I.J. & McCarty, P.L., 1965).Table 2. The composition of culture mediumComponents* Concentration (g/l)Lactose 12NH4C1 0.42(NH)2HP0 0.8KCI 0.7MgSO4.7H20 0.35Fe(NH)S0 0.2ZnSO4.7H20 0.005MnC1.4H 0.005CuSO.5H20 0.002NaB4O.10H 0.002NaMoO.2H0 0.002* All are CSA reagent grade chemicals.2.2.3 Preparation of the bacterial inoculumThe bacteria seeds used in this study were a mixed-culture of various anaerobic37bacteria dominated by acidogens. They were originally obtained from municipal sewage(lona Sewage Treatment Plant) and stored in a cheese whey substrate. Before theexperiments started, the bacterial inoculum was prepared and acclimated by the followingprocedures:1) 200 g of the bacterial culture with dense cheese whey is dispersed with ahomogenizer in 500 ml of cold water, which was previously saturated with nitrogen. Thenthe solution was quickly collected and loaded into two batch reactors (500 ml glassreactor, Fisher Scientific) (figure 2), containing 250 ml of culture medium diluted to give 4gil of lactose. Nitrogen was continuously introduced into the reactors in order to removeoxygen. The remaining solids were discarded. After 5 minutes the nitrogen supply wasstopped and the bacteria were incubated at 35 °C.During the whole experimental process, the bacteria must be protected fromoxygen.Table 3 The calcium concentrations in the feed (mgll)Reactor Run 1 Run 2 Run 3A with high calcium 100 120 230B with low calcium 0* 80 170* No calcium added to the culture medium. The actual calcium concentration is about 1.15ppm, mainly resulting from calcium in the tap water.2) When the lactose was almost completely consumed by the bacteria, half of the38bacterial suspension solution in each reactor was removed and centrifuged at 15,000 x grelative centrifugal force for 10 minutes to harvest the bacterial cells. The cells weredispersed in 250 ml of fresh culture medium with an increased substrate concentration(increase by 4 g/l of lactose) and returned into the reactors for a new cycle of cultivation.3) The above step was repeated twice. When the bacterial growth rate tends todecrease, the bacterial suspensions from the reactors were pooled together and used as theinoculum for the bioflim experiments.2.2.4 Start-up and maintenance of the reactors1). Preparation of the solid supports. The PVC slides used for all the experimentswere newly prepared in order to eliminate the errors due to the support surfaces. Theslides were cut from a 3.175 mm thick PVC sheet and have a size of 15.0 ± 1.0 by 45.0 ±0.1 millimetres. The slides’ surfaces were thoroughly cleaned in an ultrasonic bath, dilutedhydrochloric acid (100 ml concentrated HCL’l 1 distilled water) and distilled water, andthen dried at about 70 °C and carefully stored.2) Start-up of the reactors. The two biofilm reactors with 1000 ml of the culturemedia (containing 10 gIl of lactose but different concentrations of calcium) were seededwith 500 ml of the bacterial inoculum. Initially the reactors were run in batch mode. Whenthe substrate has been used up, the reactor operation was switched to continuous modewith the desired dilution rate. When the reactors reach their steady state, the PVC slideswere inserted into the reactors and the time was set as zero time.3) Maintenance of the reactor system. To ensure the proper operation of thereactor system, regular maintenance was carried out by periodically cleaning the pH39probes, temperature sensors, feed tubing and effluent outlets, and replacing the pump headtubing.sampling portwater bath inletN2 airlockthennometer 1water bath outletmagnetic stirring plate2.3 Monitoring the reactor performance and sampling‘-“It ‘Figure 2 The batch reactor for bioflim specific activity analysis40[Sampling of biofilmsj 2 - 3 of the slides (bioflims) were removed from each reactorevery 5 - 8 days for analysis for bioflim area, wet and dry biofilm biomass, bioflim carbon,bioflim specific activity, the immobilized calcium concentration, and bioflim volume. Incases where biofllms were exchanged between the two reactors in order to observe theeffect of changes in calcium concentration on the biofllm development, 1 or 2 more biofilmwere sampled at the same time.[Monitoring the reactor performance] During the experiments, the pH and temperaturein the reactors were recorded three to four times a day; the reactor effluents were sampleddaily and measured for biomass concentration. Every two days they were measured forsubstrate concentration. The effluent flow rates were also measured on a daily basis. Themorphology of the bacteria in the effluents were examined with an optical microscope andthe components of the gaseous products from the reactors were analyzed twice a week.[Sampling of feeds] The feeds were sampled every 3 - 4 days at the sampling ports andanalyzed for lactose and calcium.Calibrations or adjustments of the influent flow rates, the reactor liquid surfacelevels and the influent calcium concentrations were made whenever disagreement of any ofthese parameters with the desired experimental conditions occurred.2.4 Sample analysisUnless specified otherwise, all the chemicals used for the following analysis were41CSA reagent grade. The centrifuge used for sample preparation was the UniversalCentrifuge - model UV (ffiC).2.4.1 Turbidity of bacterial suspensionTurbidity measurement is a simple method for quick approximation of bacterialgrowth in the culture broth. Through the comparison tests, the light wavelength of 660 nmwas selected for this study because the absorbance of the samples at this wavelength had abetter linear relationship with the dry weight of the cell mass than other wavelengths suchas 420 nm. The samples were diluted to give an absorbance between 0.01-0.8 beforereadings were taken with a spectrophotometer (Baush & Lomb Spectronic 70). Distilledwater was used as the blank. The turbidity can be converted into dry biomassconcentration with the calibration curve as shown in figure 3 a.2.4.2 Wet weight and dry weight of bioflims and suspended cellsBoth the wet weights and dry weights of the biofilms were determined to directlymeasure the biofilm mass. After their surface areas were measured, the intact biofilms withtheir support PVC slides were weighed and then dried at 80 °C to a constant weight. Theweights of the wet and dried bioflims were obtained by subtracting the slides weight fromthe total weights. The dry bioflims were kept in the desiccator for analysis of TOC.For the liquid samples, the dry cell mass was determined by filtering two ml of thebacterial solution using 0.45 p.m membrane filters and drying the filtrate at less than 80°c42to a constant weigh.2.4.3 Total organic carbon (TOC)Measurement of TOC content of samples is an indirect quantitation of bioflims orsuspended bacterial mass. The TOC measurement in this study was conducted with acarbon analyzer (ASTRO 580). The principle is that the sample solution is first treatedwith phosphoric acid solution to remove the inorganic carbon-containing compounds, thenthe remaining organic components are oxidized with sodium persulfate solution andultraviolet light. The carbon dioxide produced is carried by oxygen to an infrared detector,where absorption of infrared light by the carbon dioxide generates a signal proportional tothe carbon dioxide concentration.For the biofilm samples, the dried bioflims were pretreated by dissolving them insulphuric acid solution with a pH 0.8 to make up 100 ml solution for each sample. Anultrasonic bath was employed to assist the dissolution of the biofilms. For the bacterialculture solutions, two samples were prepared: one was untreated and the other wascentrifuged at 15,000 x g for ten minutes to remove the cells. The cellular carbon wasobtained by subtracting the carbon content of the soluble organic components from theoverall carbon content of the effluents.Ethylene glycol was selected to make the standard solutions. The carbon analyzerwas operated according to the procedures specified by its manufacturer.2.4.4 Lactose43Lactose in the samples was determined by the phenol sulphuric acid method, whichis based on the colour-producing reactions of the sugars consisting of free reducinggroups with phenol and concentrated sulphuric acid (Dubois at al., 1956).Turbidity (A660)vs. Dry biomassFigure 3a Calibration Curve for Measurement of Bacterial Cell Concentration byColorimetric Method3.02.5I1.0yO.04599+3.0S42x0.00.2‘%6044Each sample was centrifuged at 15,000 x g for 10 minutes to remove solids andthen diluted to 10 -120 mg/I of lactose. One ml of the diluted solution was mixed, insequence, with one ml of 5% (by weight) phenol solution and five ml of concentratedsulphuric acid in a test tube (15 ml, Kimax). The test tube was placed in the air for tenminutes and then in a water bath at 23-30°C for another ten minutes, before its absorbanceat 480 nm was read with the Spectronic 70 spectrophotometer (Baush & Lomb). Thenthe reading was converted to the lactose concentration by using the calibration curve(figure 3b). Distilled water was used to substitute the sample solution in preparing theblank.2.4.5 Biofllm volume, density and thicknessA proportion of an intact thick bioflim was scraped into a small test tube (Kimax,diameter 4 mm) with a cap, weighed and centrifuged at 15,000 x g for 10 minutes toseparate the bioflim liquid from the solids. The surface level of the bioflim was carefullymarked on the tube wall. Then the biofilm water was withdrawn from the tube by using a1.0 ml syringe with an appropriate needle and the tube was weighed to determine thebiofilm water volume. The biofilm water was returned to the tube. A small volume ofdistilled water used to rinse the inner wall of the syringe was also injected into the tube.The tube was then dried at 80 °C to a constant weight. The dried mass in the tube wasthoroughly removed and dissolved in sulphuric acid solution to make 50 ml solution foranalysis of TOC (as described in section 2.4.4). The empty tube was cleaned and dried. Itwas refilled carefully with distilled water to the mark and weighed.Therefore, the total volume of a bioflim was determined by calculating the volume45of the refilled water from its weight and density. And based on the total volume andweight of the biofilm, the bioflim density was obtained. The bioflim water volume was alsomeasured by subtracting the weight of the bioflim solid from the total weight of thebioflim. The bioflim carbon concentration was equal to the ratio of the measured TOC tothe total volume.The average ratio of TOC/volume of several thick bioflims from the same reactorwas used as a constant coefficient to calculate the volumes and thickness of the biofilmsfor the same calcium level. The volumes of the bioflims from the same reactor wereobtained by dividing their TOC values by the average TOC/volume ratio; and their bioflimthicknesses were determined by dividing their TOC by their areas and by the averageTOC/volume ratio, or they were calculated from their volumes and areas. This techniquewill be discussed in section 3.2..2.4.6 Assay of bioflim specific activityThe bioflim specific activity was expressed as the maximum substrate consumptionrate per unit of bioflim area in the batch culture of the resuspended cells from a bioflim. Itrepresents the sum of the number of viable cells in a bioflim and the reproduction andmetabolic activity of these cells, and reflected the activity of the bioflim in situ.To determine the specific activity of a bioflim, the sampled intact bioflim after itsarea was measured was quickly transferred to and dispersed in 120 ml of the fresh culturemedium containing 5 g/l of lactose, and cultivated in a batch reactor (Kimax 125 ml glassreactor, as shown in figure 2) at 35 °C. During this process nitrogen gas was introduced toboth the culture medium and the reactor to protect the bioflim from exposure to oxygen.46The turbidity and lactose concentration of the bacterial culture in the reactor wasdetermined periodically until the bacterial growth reached the stationary phase. Then thelactose concentration and turbidity as functions of time were plotted and the maximumsubstrate consumption rate was measured from the graph (an example is shown in figure3). The biofllm specific activity was obtained by dividing this rate by the biofilm area.Lactose Standard CurveFigure 3b Standard Curve for Determination of Lactose Concentrationy.O.2E826÷148.464 x0.0 0.2 0.4 0.6 0.8 10A480472.4.7 Total calciumThe total calcium in the samples was measured with an atomic adsorptionspectrophotometer (Vedeo 220 aa/ae spectrophotometer, Thermo Jazzell Ash). When acalcium sample is aspirated in the flame and atomized, the calcium absorbs the light with acertain characteristic wavelength. The amount of light absorbed is proportional to theamount of the calcium atomized. Therefore, the calcium concentration is detected.The samples, homogenized with an ultrasonic homogenizer when necessary, wereacidified to a pH below 2.0 with concentrated hydrochloric acid. The sample solution wasthen diluted to 5-30 ppm calcium with lanthanum solution containing 58.65 grams ofLa203 and 250 ml concentrated hydrochloric acid per litre. The spectrophotometer with athree-slot Boling head was operated in the automatic mode and the operating conditionswere set as following: hollow cathode lamp for calcium; wavelength 422.7 nm, band width2.0 mm, fuel (acetylene) 5 psi., air 28 psi, current 5 mA. The standard solutions werediluted from the 1000 ppm calcium (CaC12) stock solution for atomic adsorptionspectrophotometry (Fisher Scientific). Distilled water was used as the blank and a distilledwater containing 1.5 ml concentrated nitric acid/i was used for rinsing the atomizer.2.4.8 Bioflim mineralsThe dried bioflims were first ashed at 600 °C overnight and then cooled to roomtemperature in a desiccator and weighed.482000ITime (hrs)1300080006000400020000Figure 3 A typical relation curve of cell growth and substrate consumption forbioflim specific activity analysis2.4.9 Gaseous productsThe gas samples from the reactors were analyzed for the major gaseous products,carbon dioxide and hydrogen, by using a gas chromatography (CARLE model 311) whichwas equipped with a thermal conductivity detector and a 1/8” x 20’ Paropack Q column.49The column temperature was set to 3 5°C. The flow rate of the carrier gases, helium foranalysis of carbon dioxide (and methane) and nitrogen for hydrogen, was 20-25 mI/mm at30 psi.50CHAPTER 3RESULTS AND DISCUSSIONSix different calcium concentrations were applied to the fixed-bed reactors forthree runs of experiments in this study. Each of the experiments was run for more than 70days to let the bioflim mass reach a constant level or a maximum. Some of the bioflims inRun 1 and 3 were transferred from one reactor to the other with a different input calciumconcentration after they had grown for 30 - 35 days. The measurements described in thelast chapter were made in each experiment. The results will be presented and discussed inthe following sections in order of their importance for the experimental objectives.3.1. Biofllm biomassBioflim biomass was expressed as bioflim total organic carbon (TOC). Theaccumulation of bioflim biomass for the different calcium concentrations is presented infigure 4. In the early stage of the experiments, the biofilms grew very slowly and thebioflim biomass accumulation did not show much difference for different calcium levels.After about twenty days, however, the biofllm growth speeded up and the growth ratesreached their maximum in thirty to forty days for the calcium concentrations in 0 - 120mg/l. For the higher calcium levels, the lag phase lasted longer. After the rapid growthstage, the bioflim growth tended to stop and the biofilm biomass was maintained at its51maximum level. It can be seen that the bioflim biomass accumulation follows the generalgrowth pattern of free cells.E1.5HFiguit 4.40DaysIofiIm TOC vs. Time52Importantly, the results indicate that for the range of low calcium concentration,bioffim biomass accumulation increased with increasing calcium concentration and reacheda maximum for 120 mg/I of calcium. At higher calcium concentrations, both the rate andextent of bioflim biomass accumulation decreased (figure 5). Calcium in the bulk liquidinfluences the bioflim formation and development in two ways. Firstly, it plays a role inconditioning the support and bacterial surfaces so as to effect bacterial adsorption to thesurfaces. Secondly, it is involved in the formation of the biofilm structure. It may berequired for bacterial cell growth as well, e.g., in A. xylinum, cellulose synthesis isregulated indirectly by the level of intracellular calcium ion concentration (Withfleld, C.,1988), but the calcium concentration in the bacterial cells usually is very low, e.g., i0 Min E. coil (Rosen, B.P., 1984) so that it is thought that calcium present in the tap waterwould be sufficient for the needs of bacteria. Therefore, in these experiments, where thelow pH and the presence of many other cations might offset the effect of calcium on thebacterial adsorption to the support surfaces and the biofilm accumulation for variouscalcium levels did not appear significantly different at the beginning, calcium’s role in theformation of bioflim structure was important for the development of the biofilms.Since the biofilm accumulation is mainly the result of bioflim growth anddetachment, that the presence of calcium increased in the bioflim accumulation can beattributed to two mechanisms.1. The biofilm structure was strengthened by calcium ions and the biofilms becamemore resistant to the erosion effect caused by fluid shear stress. The bacterial cell surfacesand EPS are usually negatively charged and their association with each other to form abiofilm often requires cations, mostly divalent cations to act as a bridge between thecomponents (Visser, J., 1988; Costerton, J.W., Cheng, K,J., Geesey, 0.0., et at,1987; Lam, S.S., Thompson, J.B., & Beveridge, T.J., 1993; Beveridge, T.J., 1988).53Therefore, the presence of calcium ions would facilitate the linkage of cell-polysaccharideand polysaccharide-polysaccharide. Certainly, calcium ion concentration may alsocontribute to the stability of the conformation of the polymer network in the biofilmbecause of its interactions with the secondary functional groups in the polysaccharides likeOH-, and consequently its influence on the folding of the polymers.C0ICalcium concentration in feed (mg/l)0.50.05J 2Figure 5 Biofilm biomass vs. Ca concentration542. An increased calcium concentration in the bulk liquid or calcium accumulationwithin the biofilms might create, to some extent, an environment which influences thephysiological and biochemical aspects of the cells. As a result, the synthesis ofextracellular or intracellular polymeric substances by the bioffim cells was stimulated orinhibited, a). Various metal ions including calcium have been known to be required ascofactors in polysaccharide synthesis (Larsen, B. & Haug, A., 1971). Experimentsshowed that for many bacteria, the presence of metal ions could increase the production ofexopolymers that make up the biofilms or change the exopolymer compositions and eventhe capsule morphology. For example, the polysaccharide synthesis by Enterobacteraerogenes was stimulated by calcium, magnesium and potassium ions in the culturemedium. The presence of ferrous and calcium ions enhances the polysaccharide productionin Chromobacterium violaceum (Couperwhite, I. & McCallum, M.F., 1974). Anincrease in the concentration of Cr(III) led to increased polymer production by acoryneform bacterium isolated from Cr-polluted marine sediments (Lam, S.S.,Thompson, J.B., & Beveridge, T.J., 1993). Removal of calcium from the culturemedium resulted in an increase of mannuronic acid in the alginate produced byAzotobacter vinlandii (Couperwhite, I. & McCallum, M.F., 1974; Larsen, B. & Haug,A., 1971). b). Calcium ions can increase the permeability of the cellular outer membranesof Gram-negative bacteria (Doyle, R.J., 1989) that include most of the species involvedin the anaerobic acidogenesis. The outer membrane permeabilizing effect of cold calciumion solution at 20 mM or more was described by Brass (1986) and Nikaido and Varr(1987). It is suggested that the excessive binding of calcium ions to thelipopolysaccharides which construct the surfaces of outer membranes of Gram-negativebacterial cells “freezes” the lipopolysaccharide monolayer by raising its meltingtemperature and this easily creates outer membrane cracks, through which55macromolecules can diffuse.On the other hand, the presence of too much calcium in the bioflims could damagethe environment required for maintenance of the bioflim structure or the bioflim cellularactivity and limit the bioflim development. Calcium toxicity will be examined in section 3.4where calcium concentrations in the biofilms are presented.3.2. Biofllm thicknessThe changes in biofilm thickness with time directly reflect the progression of thebiofilm development (figure 6).Because of the rough surfaces and gel-like characteristics of biofilms, thicknessmeasurement of thin bioflims remains a difficult subject in bioflim research although manymethods have been developed (Paviostathis, S.G. & Giraldo-Gomez, E., 1991). Themethods for measurement of biofilm thickness can be classified into three principalcategories, based on the principles applied: 1. by optical microscopy, 2. by means ofelectronic conductance and 3. by measurement of biofilm volumes through volumetricdisplacement. In optical microscope measurements, errors often occur as a result ofirregularities in the biofilm surfaces. These methods require a optical microscope with veryfine stage adjustments and the results should be corrected for the refractive index of thebiofllm. For the methods in the second category, specially designed probes with electroniccircuits and electrometers are required.The author contrived a device for the case of this study (see figure 7). When it isused, probe B is first placed on the substratum-biofilm interface and then probe A wasslowly inserted into the biofilm surface by turning the knob. Both of the probes have two56layers of metal coatings which are separated by insulating materials and allowed to acquiredata on two levels for a measuring point. The bodies of the probes, which formed twoelectrodes of a circuit, could be used to signal the improper placement of probe A. Themajor difficulties would be encountered in coating the probes and calibrating the device.CDaysFigiie 6. Biofilm Thickness vs. Tmi57meterU’supportknobstandcoatingmaterial(layerthickness=5-lOurn)Figure7AconcenptialdevicefordirectmeasurementofbiofilnithicknessbydeterminingcontuctanceFor the volumetric displacement methods, problems often occur in replacing anexact area of bioflim with water or reading the water volume because the biofilm is notonly very small in volume but also sticky.The method used in this study provides another technique for the estimation ofbioflim thickness. It was modified from the method used by Yu and Pinder (1991), inwhich the thickness of thick bioflims was obtained from measurement of the biofllmvolume by volumetric displacement, while thin bioflim thickness was calculated from thedensity measured from the thick biofilms, the mass and area of the thin biofilm.It was assumed that the biofllms for a given calcium concentration werehomogenous, had the same structure, the same component distribution and the samecellular activity in any portion at any time. Therefore, the carbon content per volume ofbioflim for a given calcium level could be considered to be constant, or(c’ (c (C”I—I =1—I =1—I =•=k=constant (1)i,V)1 iV)2 V}3or(C (C (CH—I =i—i =1—i =...=k=constant\SO)j S.OJ2 iS.OJ3Where:C -- the bioflim carbon (mg)V -- the biofllm volume (cm3)S -- the biofilm area (cm2)o -- the biofllm thickness (cm)k -- constant, or the biofllm carbon concentration for a given calcium level (mg/cm3)The subscript number stands for the bioflim samples at different times.59In order to obtain the value of the coefficient k, several pieces of intact thickbiofllms for the same calcium level were sampled and measured for biofilm volume andcarbon content. The process is briefly described as follows. For details, refer to section2.4.5 in Chapter 2. A small mass from a piece of biofilm was scraped into a small test tubewith a diameter of 4 millimetres and centrifuged at 15,000 x g to draw all of the bioflimmass into the bottom of the tube. Then the test tube with the biofilm mass was weighed todetermine the biofilm density, and the total volume of the biofilm was determined byreplacing the biofllm mass with the same volume of water and calculating the volume ofthe water from its weight and density. The biofilm mass was analyzed for total organiccarbon after it was withdrawn from the test tube. The average ratio of the bioflim carbonto the biofllm volume of these thick biofllms was then used as the k value for the givencalcium concentration.For the biofilms with known carbon contents and corresponding areas, theirthickness could be obtained by performing the following calculation:(2)Where:—— bioflim carbon per unit of bioflim area (mg / cm3)O and k are the same as in equation (1).Compared with the previous technique used by Yu and Pinder, this technique hasthe advantage that it eliminated the need to collect the mass of an exact area of biofilmduring the measurement of volumes of thick biofilms in small test tubes, which was a great60difficulty in practice.The k value in the above equations is a reflection of change in the biomassconcentration of biofilms. As shown in figure 8, the relationship between the carbonconcentration of the biofilms and the input calcium had a similar trend to that of thebioflim mass and the calcium, although changes in the bioflim carbon concentration at lowcalcium concentrations are less striking than that in the biofllm mass. This indicates thatthe bacterial biofilms tended to build a structure with denser organic mass when the inputcalcium was increased, but when the calcium concentration was beyond a certain level, theorganic mass concentration of the biofilms declined dramatically.It is not known whether this was because calcium influenced the polysaccharideproduction in the bacteria, altered the biofilm structure, or because the build-up of bioflimmass increased the resistance to diffusion of the substrate, nutrients, electron acceptorsand the bacterial metabolic products between the bulk liquid and the cells in the lowerportion of the biofilms and resulted in changes in the physiological states of these cells.Possibly, both of these causes existed, that is, the presence of calcium stimulatedproduction of the polymers by the bacteria, or calcium interacted with exopolymers in thebioflims and caused a change in the physical state of the polysaccharides ( such as gelformation in the case of alginic acid -- Geesey, G.G. & Jang, L., 1989), which furtherinduced the bacterial cells to synthesize polymers. A simulation study on naturallyimmobilized Anabaena azollae cells suggested that confinement of cells in a restrictedspace could induce the development of a mucilaginous envelope and attachmentmechanisms. This in turn would lead to changes in the physiological and biochemicalbehaviours of the cells. Another possibility is that the development of the polysaccharidemucilage has an effect on the water activity and as a consequence on the metabolism of61the cells (Hamer, G., 1990).I—• I140 0 0 200 220 240Calcium concentration (nig”l)Figuff 8. lioflim caiton concentration vs. calcium concentration in feed138646062Here it is also worth mentioning the observation by Haug and Larsen (Larsen, B.& Haug, A., 1971) that acetate could stimulate •the production of extracellularpolysaccharide in Azotobacter vinilandii. For the case of acidogenesis of lactose byanaerobic bacteria in this study, acetate is one of the major products. This could be thebasis for another mechanism for the biofilm biomass production associated with calcium.3.3. Bioflim specific activityThe biofilm specific activity, expressed as maximum substrate consumption rateper unit of dry bioflim mass when the cells were cultivated in discrete states (see section2.4.6 in chapter 2), is the sum of the activities of biofllm cellular reproduction andmetabolism. It reflects the viability of bacterial cells in a bioflim, or the number of viablecells and their activity in a biofilm. The biofilm specific activity as a function of calciumconcentration is presented in figure 9. It can be seen that the specific activity of the bioflimtends to decrease with an increase in calcium concentration in the medium although itappears quite stable for a low calcium concentration range, indicating that the biofilms athigh calcium concentrations had fewer viable cells or a lower cellular metabolic activity.The bioflims for lower calcium levels reached their highest specific activity earlier than dobiofilms for higher calcium levels (figure 10), indicating that the cells in the bioflims at thehigh calcium concentration took more time to recover their cellular activity.Compared with the results on biofilm mass accumulation (see 3.1) and biomassconcentration within bioflims (see 3.2), this implies that at a low calcium concentration,loss of the biofilm specific activity resulted from the increasing transport resistance due tothe increase in the bioflim thickness and from the lower viable cell count or lower water63activity because of the higher polymer concentration. For the high calcium concentrationrange, the biofilm specific activity decreased due to the increase in calcium inhibition tothe biofilm cellular activity or enzyme activity (toxic effects), or damages to the bacterialcells caused by calcium accumulation within the biofilms.14000020000111)0000800006000040000020000013) 200Calcium concentration in feed ( mg/I)Firgure 9. Biofilm specific activity vs. calcium concentration64a)IIpIn bioflims, the bacteria cells rely entirely on diffusion from their immediate localenvironment to obtain nutrients and to disperse metabolic waste products. Hence, watercontent in biofilms is very important for carrying necessary solutes to the cells. Moreover,water is also required by the bacteria to maintain cellular integrity and proper metabolicfunctioning (Beveridge, T.J., 1988). Therefore, the higher polymer accumulation both intotal amount and in concentration would either deny the access of nutrients to the bacterialTime (diys)FigurelO. Biofilm specific activity vs. Time65cells or decrease the water activity, resulting in changed metabolic activity and productformation rates of the cells in bioflims. It was reported that the presence of polymers evenat a low level had a substantial effect on biochemical reactions which are water-dependent(Brouers, M., Shi, D.J. & Hall, D.O., 1990).ti), )C) )C.,)For similar reasons, too high a calcium concentration would seriously influence the0.05 0.10 0.15 0.20 0.25 0.30 O.3.Biofilm thickness (cm)Figure 11. Biofilm specific activity vs. thickness66bacterial growth and metabolism in biofllms. Besides, calcium ions may alter the surfacepermeability of the bacterial cells (see 3.1). A large quantity of calcium immobilized in thebioflims may cause autolysis of the bacterial cells as well. This was suggested by theobservations of Harvey et. al.(1984), in which the mature methanogenic bacterial biofllmsformed on the polyester supports, contained extensive deposits of electron-dense, calcium-and phosphorus-enriched material completely entrapped cells. The trapped cells appearedto autolyse, and many were partially degraded. They postulated that further impregnationof the matrix with minerals and apparent cell death may eventually have a deleteriouseffect on the methanogenic activity of the biofilm.The mechanisms of metal interactions with bacterial cells and polysaccharides willbe further discussed in the next sections (see 3.4 and 3.5).From the curve of bioflim specific activity and thickness (figure 11), it can be seenthat biofllms with a thickness less than 0.5 millimetres have the highest specific activity.Since the overall efficiency of a fixed film reactor depends on the mass of bioflim and thebiofllm specific activity, the substrate removal rate per unit of biofllm surface area wasplotted as a function of calcium concentration in the feed to find the optimum inputcalcium level for acidogenic biofilms (figure 12). It can be seen that the optimum calciumconcentration is 100 - 120 mg/i.3.4. Immobilized calciumAs shown in figure 13, the total calcium concentration in the biofllm wasproportional to the calcium concentration in the external medium. Calcium accumulationwithin bioflims mostly resulted from the interaction of calcium ions with the670a)08a)Calcium concentration in feed (mg/I)Figure 12 Biofilm activity vs. Calcium concentration in feedexopolysaccharide polymers and bacterial surfaces because calcium concentration inbacterial cells normally is very low. Calcium accumulation is a non-specific process, whichis driven by the calcium ion gradient from the bulk liquid phase into the bioflims.*45,4035.30’2520’1513-5-0 501130 250Non-specific binding of metal ions to the cell wall, capsule and extracellular slime68layer is one of the two main mechanisms of metal accumulation by bacteria. The bacterialcapsules are composed of the exopolymers or polysaccharides that are intimatelyassociated with the cells, while the slime layers are formed by the polysaccharides that areloosely associated with the cells (Withfield, C., 1988). In bacterial bioflims, thesepolysaccharide polymers ‘knit’ the extensive network that embraces all the othercomponents of the bioflims. They create a buffer zone between the surfaces of cells andtheir external environment, which is developed in response to the external environmentalconditions. When the nutrient concentration in the surrounding environment is low, theyextract and accumulate nutrients, especially metal ions from the environment so that thebacteria would gain a better control over concentrations of nutrients that actually reachthe cell surfaces, and when metals exist at toxic levels in the environment, they function asa barrier to protect the cells (Lam, S. S., Thompson, 3. B., & Beveridge, T.J., 1993).Capsules and slimes are the first structures encountered by metal ions when theyare present around the cells. Due to their chemical nature, they are ideal cation scavengers,resembling cation exchange resins, and can accumulate large quantities of metal ions. Forexample, a capsule-forming bacteria, Zooloea ramigera was found to contain up to 25%of metals by weight after growing in sewage sludge (Geesey, G.G. & Jang, L., 1989).The chemical compositions of capsular and slime polysaccharides are usually similar whenboth capsules and slimes are produced (Geesey, G.G. & Jang, L., 1989). Thesepolysaccharides contain uronic acids and other substituted sugars that possess acidicfunctional groups important for metal binding, mainly carboxyl and hydroxyl groups. Ofthe two chemical groups, carboxyl groups are the most active. Most polysaccharides arenegatively charged, and metals are usually bound by cross-bridging between anionicgroups. This neutralization of charge by the metal ions often results in coprecipitation ofthe metal-polymer composite resulting in fioc formation (Geesey, G.G. & Jang, L.,691989). This is considered as an important mechanism of metal sedimentation from the fluidphase in natural aqueous environments, brought about by organisms. In case of unchargedpolysaccharides, weak electrostatic interaction between metals and hydroxyl groupsbecome important. The affinity of uncharged polysaccharides for metal ions generallydecreases with increasing radius of the hydrated metal ions. In general, an acid-basereaction is involved which results in the liberation of protons. Such reactions aresignificant in the biocorrosion of metal surfaces (Geesey, G.G. & Jang, L., 1989).0.045-0.040-0.035-‘— 0.030-0.025-0.020-0.015-0.010-0.005-0.000 I0 50 250Figure 13. Biofilm calcitin content vs. caliciurn concentmtion in cilture methinBacterial cell walls tend to have a net negative charge at circumneutral pH mainlyCa concen. in the feed (mg4)20070because of the acidic groups of their polymer components. In some cases, the bacteria’shigh capacity to accumulate metal is attributed to this charge character (Beveridge, T.J.,1984). For Gram-positive bacteria, the prime site of metal binding in the cell wall seems tobe the carboxyl residues of peptidoglycan and teichuronic acids, and the phosphorylgroups of teichoic acids. The contribution of each group to overall metal binding capacityof the cell depends on the amount of each polymer present, which varies with the strainsand their culture conditions (Doyle, R.J., 1989). For Gram-negative bacteria, the majorfunctional groups of the cell wall responsible for metal binding, in sequence of importanceare phosphoryl groups and carboxyl groups of lipopolysaccharide molecules in the outermembrane and the same groups in the peptidoglycan layer (Lam, S.S., Thompson, J.B.,& Beveridge, T.J., 1993). Generally, Gram-positive cell walls bind more metal than dogram-negative walls (Beveridge, T.J., 1984; Lam, S.S., Thompson, J.B., & Beveridge,T.J., 1993). In both cases, the quantities of bound metal are greater than the number ofavailable anionic sites in the walls. To explain this phenomenon, a two-step mechanism formetal deposition in the bacterial wall was proposed by Beveridge and Murry (Beveridge,T.J., 1984). The first step involves the stoichiometric interaction between metal ions andactive sites within the wall. Next, this initial bound metal would act as nucleation sites forthe deposition of more metal from the solution. The metal aggregates grow within the walluntil they are physically limited by the available space within the wall fabric. In this way,metals deposited in the wall are not easily redissolved by water or replaced by protons orother metal ions. Therefore, the matrix of the bacterial wall provides a special environmentfor the nucleation and growth of metal aggregates.Binding of calcium ions to the exopolymers and cells in the biofilms wouldinfluence the exopolymer production and the physical state of the exopolymers (seesection 3.1 and 3.2), and the polymers in turn will affect the calcium adsorption to the71bioflims. The changes in the composition and behaviour of the bioflims for differentcalcium concentrations reflect the results of the interactions of calcium ions with the cellsand exopolymers in the biofllms.From figure 13, we can find that the calcium concentrations in the bioflims weremore than 10 times greater than were present in the feeds. They ranged from less than 1 tohigher than 4 g/l. The data on calcium toxicity for the acidogenic bacteria, both for thesuspended cells or for the immobilized cells, are not available. But according to Kugelmanand McCarty (1965), the optimum calcium concentration for methanation of acedic acidwas 0.005 M (0.2 g/l) and the upper limit was from 0.05 M to 0.125 M (2.00-5.00 g/l),which might be higher, depending on the antagonist cations present in the culture medium,the method of cation introduction, the organic loading, and the biological solid retentiontime (Kugelman, I.J. & McCarty, P.L., 1965). It was also reported (Hamer, G., 1990)that for expanded/fluidized bed reactors, wastes containing high levels of calcium areparticularly difficult to treat. Waste with 2.5 g/l of calcium were effectively treated onlaboratory scale. However, after 150 days calcite precipitates were observed to be about30% of the total weight of the sand/biomass particles (Jordening, et al., 1988). Greaterthan 90% of the calcium in a waste containing 0.9 - 3.0 g/l calcium was retained in a10-litre laboratory-scale reactor. The accumulation of calcium resulted in increasedparticle density, loss of fluidization, clogging and 40% dead space (Vogel and Winter,1988). Based on these relevant facts and the results from this study, as indicated by themeasured bioflim specific activity and biomass accumulation (see section 3.1 and 3.3), wepostulate that an immobilized calcium higher than 2 gIl in the bioflims would imposed asignificant restraint to the cellular activity of the biofilms.It is interesting that EDX analysis and SEM analysis of the dried biofilm samplesshowed that calcium was distributed quite evenly on the biofilms, despite the earlier72anticipation that a non-uniform calcium distribution on the bioflims would occur and largecalcium deposits might exist in the bioflims. Because the calcium content of the driedbiofilms was not high enough to meet the minimum concentration level requirement forthe SEM, the imagine of calcium distribution on the biofilms could not be produced.However, the EDX graph for calcium indicated that there was a even calcium distributionin the biofilms. This perhaps can be attributed to the low pH environment for bioflimdevelopment which facilitated the dissolution of any calcium deposit. The EDX analyticalresults do not indicate whether calcium distribution was also uniform in depth in thebiofilms.3.5. Influence of calcium fluctuationThirty five days into the runs some of the bioflims were switched from one reactorto the other with a different calcium concentration in the influent. By then the bioflims hadformed a relatively thick, uniform layer over the submerged area of the plastic slides. Thesubsequent progression of the biofllm biomass development is shown in figure 14. Onlythe bioflims transferred from the reactor with 100 mgIl of calcium to that without theaddition of calcium showed a sharp decrease in biomass. The other transferred biofilmsshowed little difference in biomass growth from those which remained in the originalenvironments. That the bioflims moved from 0 to near optimum ( 100 mg/i ) calciumconcentration did not show an improved accumulation of biomass indicates that, perhaps alack of calcium during biomass attachment to the slide surface causes a structurally weakbiofilm which can’t support development of further layers.73C.)H80DaysFigure 14. Influence in biomass progression of changes in calciumconcentration during biofilm development0 20 40 6074_10000-8000-E 6000s—’ 4000-2000-0‘-j 1I ‘ I ‘ I ‘ I • I • I •0 10 20 30. 40 50 60 70 80Daysa) in the reactor without addition of Ca to feed10000-8000--‘ 6000-c4ooo0 2000-___—0• • • •0 10 20 30 40 50 60 70 80Daysb) in the reactor with 100 mg/i of Ca in feedFigure 15. Biomass concentration in the effluent751- 11111111 • I•I’I’I’I’I’I’I0 10 20 30 40 50 60 70 80 90 100 110 120Daysc) in the reactor with 170 mg/i of Ca in feed‘—, 3-2-ii___Z0—• • I ‘ I • I • I ‘ I ‘ I • I • I ‘ I • I •0 10 20 30 40 50 60 70 80 90 100 110 120Daysd) in the reactor with 230 mg/i of Ca in feedFigure 15. Biomass concentration in the effluent76On the other hand, it was observed that serious sloughing occurred from thesurfaces of the bioflims started at 100 mg/I of calcium after they were transferred to thereactor without added calcium. This was further proven by the fluctuation in thesuspended biomass concentration in the effluent (figure 15 a.) after the switch in samples.It is postulated that calcium within the biofllms which had a high calcium concentration,trends to leave the biofllms and enter the external environment which has a much lowercalcium level and this would decrease the stability of biofilm structure and greatly speedup biofilm detachment due to the concentration shock.In a study on the effects of nutrient limitation on bioflim sloughing with theobligate aerobe Pseudomonasputida, Applegate and Bryers (1990) found that the bioflimswith higher extracellular polymer content and bound calcium exhibit a much lower shearremoval rate. However, these biofllms always experience catastrophic sloughing events.They attributed the characters of reduced shear removal and the susceptibility to sloughingin these biofilms to their denser, more rigid crystalline structure brought about byexcessive polymer production and concomitant binding of calcium.3.6. Biofllm compositions and densityThe compositions of the bioflims are listed in table 4 . It can be seen that thebiofilms contained 87-96% of water and 4-13% of dry mass. Compared with somebiofilms formed in aquatic environments or industrial fields, which may contain up to 99%water by weight (Geesey, G.G. & Jang, L., 1989), the bioflims produced in this studyhave higher solid contents. This is probably because they were cultivated in the nutrient-enriched culture medium.77As illustrated in figure 16, when calcium concentration in the feed was raised, thewater content of the bioflims decreased and the total dry mass of the bioflims increased.Table 4. The composition of dried bioflimsCa in feeds Water Dry mass Ash Organic(mg/i) % % % %80.00 95.73 4.27 1.45 2.82120.00 94.89 5.11 2.14 2.97170.00 91.16 8.84 6.04 2.80230.00 87.40 12.60 9.81 2.79However, in the dry materials, the proportion of minerals increased significantly while thatof organic materials slightly decreased. This indicates that the presence of calciumWater 100%Organic 60%70%________60%50%40%30%20%10%______0%80 120 170 230Calcium in feeds (mg/I)Figure 16. Calcium concentration vs. Biofilm compositionincreases the dry mass of the biofilms mainly by increasing the concentration of minerals in78the bioflims. The increased mineral content is very likely the result of more calciumtrapped in the biofilm. The density of the biofilms increased with increasing calciumconcentration in the feeds (figure 17), reflecting the changes in the biofllm compositions.At this point, we can probably narrow down the reasons for changes in the biofilmspecific activity and the bioflim mass accumulation with calcium concentration. For thehigh calcium concentrations, a large amount of mineral deposits within the biofilms alongIFigure 17. IofiIm density vs. Calcium Concentration in feedwith the significant decrease in the bioflim water content would cause an inhibition of the100 150Caconentration infeed(mg’l)79cellular metabolism and even damages to the cells, resulting in a lower biofllm specificactivity and biofllm mass accumulation. When the calcium concentrations were lower, thestress to the cells that was caused by accumulation of minerals within the bioflims wasless, if the calcium did not promote the cellular reproduction and exopolymer production.Therefore, the bioflims showed a higher mass accumulation and when the cells in thebiofllms were dispersed in the fresh culture medium, they would exhibit a higher specificactivity.A more important mechanism for the biofllm accumulation at low calciumconcentrations is likely that with the increase of the calcium concentration, more boundcalcium in the biofilms led to a stronger biofilm structure, resulting in a smaller loss of thebiofilm mass. This explanation is also supported by the results reported by Turakhia andCharacklis (1988). In their study of the effect of calcium on the bioflims of marine bacteriaP. aeruginosa in the RotoTorque reactor, they found that in the calcium concentrationrange from 0.4 to 50 mg/l, increasing calcium concentration in the growth mediumsignificantly increases the bioflim accumulation; the cellular activity, indicated by specificgrowth rate and biomass yield, was the same as that measured in a chemostat and was notinfluenced by the calcium concentration; extracellular polymer production rate in thebiofllm was unaffected by the calcium concentration but was higher than that determinedin a chemostat. They did not explain clearly why the biofilm accumulation increased in thepresence of calcium. But they also reported that calcium in the biofilm increasedproportionally to the calcium concentration in the culture medium and the cohesiveness ofthe biofllm, as indicated by a lower relative detachment rate, was increased.80CHAPTER 4CONCLUSIONS AND RECOMMENDATIONS4.1 Conclusions1. The presence of calcium in the growth medium increases both the mass accumulationrate and ultimate total mass of anaerobic acidogenic biofllm when the calciumconcentration in the feeds is in the range from 0 to 120 mg/l. For a higher calciumconcentration, the biofilm accumulation may decline.2. The specific activity of the acidogenic biofilm decreases with increasing calciumconcentration in the medium. The bioflim thickness for the highest specific activity is lessthan 0.5 millimetres. The optimum calcium concentration for the substrate consumptionrate/unit area bioflim is from 100 to 120 mg/i.3. Calcium accumulation in the biofilms is proportional to the calcium level in the liquidphase.4. Increasing calcium concentration in the culture medium increases the stability of thebiofilm structure when the calcium concentration is not higher than the optimum level.814.2 Recommendations1. Further study on the relationship between external calcium concentration and EPS andthe cellular mass in the biofilms is recommended for a better understanding of themechanism of the higher acidogenic bioflim mass accumulation promoted by calcium.Comparison experiments with suspended cells would be useftul for clarif,ring theexperimental results with the immobilized cells.2. For the same purpose, a farther study on the effect of calcium on the substrate andcalcium transport into the biofilms is also recommended. A study on the ultrastructure ofthe bioflims by utilization of an electronic microscopy may be important for this study.3. Utilization of a pure culture of the acidogenic bacteria in studies on bioflims would bebeneficial for better control of the experiments.4. Another design of the experimental system for the study of anaerobic bioflims ispresented in figure 16. The author believes that it will provide the following advantagesover the current reactor system:- avoid the measurement of each individual biofilm area because there is no headspace in the biofilm chamber.- make biofilm sampling easier and better because the slide bases would be made ofthe material similar to that for the chamber wall or other hard organic materials so that itwould not become deformed even after being used for a long time and would beconvenient for inserting and taking out the biofilm supports. The screw threads on thebases would facilitate the installation and removal of the support assemblies.82Figure 18. An alternative design of the experimantal system for study of anaerobic biofilmsNitrogenBiofflm SamplingPortsFeedbioflim support B/Sludge83- simp1if,’ the reactor control and maintenance greatly, because control of thereactor liquid surface level would be much easier and would no longer influence thebioflim areas; removing the sludge and cleaning the reactor would become simpler;preparation of the microbial inoculum can be made with the same fermenter.- facilitate the study of the effects of the radial flow rates on the biofilms atdifferent positions of the cross section of the chamber.Besides, the chamber can be made removable and can be easily replaced with anotherchamber which may have a different length or diameter to meet the needs of theexperiments.84REFERENCESApplegate, D.H. & Bryers, J.D. (1990). Bacterial bioflim sloughing: nutrientlimitation effects. In J.A.M. De Bont, I. Visser, B. Mattiasson & J. Tramper (Eds.).Physiology of immobilized cells (pp. 87-95). 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Ph.D. dissertation, U of BritishColumbia, Vancouver, B.C., Canada93AppendixRawExperimentalDataBioflimTOCmgfbiofilmTimeatatfrom100from0TimeCa120Ca80Timeatatfrom230from170daysCa100Ca0to0to100daysdaysCa230Ca=170to170to23000.00000.00000.00000.00000.00.00000.00000.00.00000.00000.00000.000080.11830.00000.11830.00007.41.58501.47858.02.80001.30002.80001.3000110.09181.88160.09191.881413.41.67171.836616.92.30002.20002.30002.2000125.722013.38455.721813.384622.36.64692.128223.75.76001.90005.76001.9000122.78929.21802.78919.217927.97.59902.029336.66.400015.33406.400015.3340180.40821.67670.23941.676936.113.481541.331642.84.500717.06004.500717.06002619.904217.236519.904117.236544.046.84563.773054.85.735518.00005.735518.00002923.893733.674523.893533.674751.088.739325.143665.827.186613.291551.254550.17983239,210648.257039.210648.257057.4107.400046.685374.919.990222.756627.726840.30103650.422742.346553.172230.569763.392.722032.930083.040.539738.383878.446547.98884172.494126.928229.527936.020363.979.476043.402688.941.050540.757164.821543.31164577.409035.548510.004338.851367.2103.538164.502196.515.686583.896418.160215.62444983.088038.218814.674536.725368.3108.384541.1213104.773.3807198.485476.285741.06725287.139721.08276.240330.076369.4137.203584.95205664.608526.997713.321123.10145990.370142.051315.340665.4096Li.DryBiofilmMassgTimeCa=120Ca=80TimeCa=230Ca170from170tofrom230to230170daysdays000.00000.00000.00.00000.00000.00000.000070.00160.00068.00.00460.01790.01797.9358130.00240.001516.90.01370.01360.013616.8685220.00430.002023.70.01540.01700.017023.6968280.00220.000036.60.02640.01040.010436.5674360.00670.003842.80.00980.01580.015842.7869440.01450.006054.80.01010.04120.041254.7683510.02110.008265.80.03260.07990.079965.6792570.03530.018074.90.04470.07770.077774.7318630.03430.019483.00.04720.04820.048282.8838630.45620.333488.90.01640.01740.017488.8412670.03490.022596.50.01190.01110.01110.0130680.53540.3354104.70.02050.01620.01620.0209700.03440.0248WetBioflimMassgTimeatCa120atCa80TimeatCa=230atCa170daysdays00.00000.00000.00.00000.000070.04010.04868.00.09430.0502130.06010.054916.90.06710.0734220.07280.021123.70.28680.0907280.05500.019136.60.08600.2848360.21790.113342.80.20930.1958440.29150.074154.80.19110.2163510.38620.186165.80.23700.0811570.48620.194374.90.19420.1750630.64330.301583.00.25380.19826388.90.28310.2419670.55910.332796.50.21300.248668104.70.42330.4472700.58680.4417AverageratioofTOC/bioflimvolumeCalevelSampleNo.DensityWetWeightMeasuredVolumeTOCTOC/biofilmvolumetimeppmg/mlmlmgmg/mm3days11.02160.48640.47610.35480.745221.03570.39830.38460.31670.823631.02730.53410.51990.40040.770180Average1.02820.47290.46020.35730.77963611.03740.11670.11250.09640.856921.04070.10320.09920.09080.915431.05120.06940.06600.06140.9300120Average1.04310.09640.09260.08290.90083611.05280.13760.13070.06320,483821.05900.15740.14860.09550.642231.06130.14500.13660.08210.6012170Average1.05770.14670.13870.08030.57573811.06300.16770.15780.05120.324521.04860.19630.18720.07590.405231.06270.18920.17800.06190.3478230Average1.05810.18440.17430.06300.35923800.000.0084.664.27114.144.39123.974.76124.894.63185.174.85264.754.68294.924.59324.685.07364.895.26415.125.24455.085.29495.345.18525.445.07565.155.39595.525.460.000.004.664.274.144.393.974.764.894.635.174.854.754.684.924.594.685.074.895.265.515.345.265.034.965.265.275.395.135.095.505.460.00.000.007.43.984.3513.44.353.9822.34.283.6827.94.503.9036.14.955.1044.04.503.9051,05.254.9557.45.255.2563.35.635.2563.95.555.5567.25.555.5568.35.405.4069.45.555.550.00.000.008.04.054.5016.94.764.0223.74.764.7636.64.804.8042.84.804.9554.84.954.9565.84.954.8074.94.954.9583.04.954.9588.95.105.1096.55.105.25104.75.105.10BioflimSurfaceAreacm2Timeatatfrom100from0TimeatatCa=80Timeatatfrom230from170daysCa=100Ca=0to0to100daysCa120daysCa230Ca170to170to230000.004.054.764.764.804.804.954.954.954.955.105.105.100.004.504.024.764.804.954.954.954.954.95 5.104.954.80BioflimthicknessandvolumeBioflimThickness(cm)TimeatatTimeatatCa=120Ca=80Ca=230Ca=170DaysDays0000007.39580.00440.00447.95830.01440.003813.3960.00430.005916.8960.01010.007122.2780.01730.007423.7290.02530.005227.8820.01870.006736.6040.02780.041636.0970.03020.103942.8130.01960.044944.0490.11560.012454.820.02420.047451.0310.18760.065265.7920.21620.132157.3540.22710.114174.8540.1170.106163.2570.1830.080582.9790.3310.126363.8750.1590.100388.8750.26540.110667.2290.20710.149196.5420.07440.208268.2920.22280.0977104.730.31240.50769.4170.27440.1963BioflimVolume(cm3)TimeatatTimeatatCa120Ca=80Ca230Ca170DaysDays0000007.39580.01760.0197.95830.05850.016913.3960.01860.023616.8960.0480.028722.2780.07380.027323.7290.12030.024827.8820.08440.02636.6040.13370.199836.0970.14970.530142.8130.0940.222344.0490.52010.048454.820.11980.234551.0310.98510.322565.7921.07040.633957.3541.19230.598874.8540.5790.52563.2571.02940.422482.9791.63820.625263.8750.88230.556788.8751.35370.564367.2291.14940.827396.5420.37921.09368.2921.20320.5274104.731.59312.585869.4171.52321.0896C CBioflimTOCPer UnitAreamg/mm2Timeatatfrom100from0TimeatatdaysC100Ca=0to0to100daysCa=120Ca=8000.00000.00000.00000.00000.00.00000.000080.00250.00000.00250.00007.40.03990.0340110.00220.04290.00220.042913.40.03840.0462120.14410.28100.14410.281022.30.15550.0579180.05710.19910.05700.199027.90.16890.0520260.00790.03460.00460.034636.10.27240.8104290.41890.36840.41890.368444.01.04100.0967320.48590.73320.48590.733251.01.69030.5080360.83870.95220.83870.952257.42.04570.8892411.03110.80481.08740.581063.31.64840.6272451.41670.51430.53630.674363.91.43200.7820491.52260.67260.19020.772767.21.86561.1622521.55680.73810.29560.698768.32.00710.7615561.60100.41620.11840.557669.42.47211.5307591.25360.50070.25990.4541631.63710.77000.27891.1989670.51740.2659700.6312(continued)Timeatatfrom230from170daysCa=230Ca=170to170to2300.00.00000.00000.00000.00008.00.03460.01440.03460.014416.90.02420.02740.02420.027423.70.06060.02000.06060.020036.60.06670.15970.06670.159742.80.04690.17230.04690.172354.80.05790.18180.05790.181865.80.51770.50690.27460.138574.90.28010.40710.20190.229983.00.79240.48470.40950.387788.90.63550,42460.40250.399696.50.17800.79900.15380.1578104.70.74791.94590.71940.4278BioflimSpecificActivityAssayCTimeInitialbioflimLactoseconcentrationTimeintervalLactoseconsumptionSpecificactivitydrymassdropratedaysmg/ihrsmg/IJhrmg/lIhrIgatCa80mg/i000.000.000.000.0070.00163691.0022.00167.77104857.95130.0024552L0022.00250.95104564.39220.00436195.0023.70261.3960788.93280.00223850.0027.30141.0364102.56440.01451009.0013.2076.445271.68510.0211477.0021.0022.711076.51570.0353239.0014.8016.15457.47630.03431072.0020.2053.071547.21atCa120mg/i000.000.000.000.0070.00063264.0022.00148.36247272.73130.00155664.0022.00257.45171636.36220.0027784.0023.70328.44164219.41280.0054210.0027.30154.2130842.49440.006865.0013.2065.5310921.72510.0082477.0021.0022.712770.03570.018120.0014.808.11450.45630.0194714.0020.2035.351821.99(continued)TimeInitialbioflimLactoseconcentrationTimeintervalLactoseconsumptionSpecfficactivitydrymassdropratedaysmg/ihrsmg/I/hrmg/IIhr/gCa=170mg/i00000.000.0080.004614523.56.171341.35170.0137148642.31168.80240.015410392639.962594.91370.026450451.179.85373.09430.009889123.8337.393815.29550.0101418716.45254.5325200.87660.0326205610205.606306.75750.0447121819.961.211369.26830.04723761328.92612.78890.016453525.8320.711262.95970.011936218.4719.601647.00atCa=230mg/i00.0000000.000.0080.017911923.55.06282.90170.0136352645.50404.41240.01711882645.692687.78370.01049052437.713625.80430.015877223.8332.402050.39550.041218417263.006383.50660.0799124410124.401556.95750.0777177322.578.801014.16830.048246812.5637.26773.05890.017485220.3141.952410.91970.011151021.3223.922155.06CellandsubstrateconcentrationineffluentCellConcentrationTOC(mg/i)TimeCa=120Ca80TimeCa230Ca170daysdays-8498.3521.90----0493.9759.97.958330.034570.01444610318316.89580.024190.027361226828423.72910.060570.0199826454.38274.536.60410.066670.1597333888.24726.442.81250.046880.172324160062854.84270.057930.1818245746.441115.965.81130.517720.5068749655.88761.0574.86720.280070.4070852624.1270482.96850.792390.4847456691.38398.3388.85550.635510.4246259695.25383.3796.55360.178040.7990163704.71747.79104.7040.74791.9459470831.76728.84771137.3775.5885763.53877.26Lactoseconcentration(mg/i)TimeCa=120Ca=80TimeCa=270Ca=170daysdays-989.580.70-----4855.7274.57.949699501.568467.921396.8324.916.90565131.852060.376118.355.323.74831055.723305.66126155.336.62836878.731992.451464.97442.81715296.787584.9019303547854.84275138.434030.1821504.5151065.81133615.873532.07261106540.574.8672430.5394305.39321137.61991.882.96852444.662060.37341689.31672.388.8555189.7012286.79401124.4863.896.55364123.944866.25451780.13873.2104.7043575.451245.2852702.24115.859720.1477.8C cjiCalciumwithinBioflimsatCa=120atCa=80Timetotal CaCa/areaCa/volumetotal CaCa/areaCa/volumeDaysmgmg/cm2mg/cm3mgmg/cm2mg/cm300000007.30.150.00190.426240.20.00230.5273100713.30.250.00290.673530.30.00380.6367643722.20.450.00530.304920.30.00410.5495115427.80.30.00330.177810.30.00380.5762979636.10.550.00560.183740.50.00490.047158544.00.50.00560.048070.10.00130.1033207651.00.90.00860.045680.20.0020.0310080657.32.20.0210.092260.60.00570.0501005963.22.20.01960.106860.60.00570.0710283363.82.10.01890.119010.80.00720.071853367.22.40.02160.10440.60.00540.0362618168.32.10.01940.087270.40.00370.0379197369.42.150.01940.070580.50.00450.02294397(continued)CatCa=230atCa=170from230from170to170to230TimetotaiCa)Ca/areaCa/volumetotalCaCa/areaCa/volumetotalCatotalCadaysmgmg/cm2mg/cm3mgmg/cm2mg/cm3mgmg0000000007.95830.30.00370.25650.20.00220.59040.30.216.8960.20.00210.20820.20.00250.34890.20.223.7290.40.00420.16630.30.00320.6060.40.336.6040.20.00210.07480.60.00630.15020.20.642.8132.30.0241.22351.50.01520.33742.31.554.822.40.02421.00193.70.03740.78892.43.765.7923.10.03130.14481.030.01070.08120.313.7374.8548.290.08370.71590.690.0070.06570.839.5382.9793.850.03890.11751.550.01570.1241.463.7588.8752.390.02340.08831.230.01210.1091.414.196.5423.220.03160.42451.150.0110.05262.133.53

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