UBC Faculty Research and Publications

Sustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled… Westman, Johan O; Wang, Ruifei; Novy, Vera; Franzén, Carl J Sep 12, 2017

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-13068_2017_Article_893.pdf [ 1.68MB ]
JSON: 52383-1.0366872.json
JSON-LD: 52383-1.0366872-ld.json
RDF/XML (Pretty): 52383-1.0366872-rdf.xml
RDF/JSON: 52383-1.0366872-rdf.json
Turtle: 52383-1.0366872-turtle.txt
N-Triples: 52383-1.0366872-rdf-ntriples.txt
Original Record: 52383-1.0366872-source.json
Full Text

Full Text

Westman et al. Biotechnol Biofuels  (2017) 10:213 DOI 10.1186/s13068-017-0893-yRESEARCHSustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled multifeed simultaneous saccharification and co-fermentation of wheat strawJohan O. Westman1,2† , Ruifei Wang1†, Vera Novy1,3,4  and Carl Johan Franzén1* Abstract Background: Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fer-mentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw.Results: More than 50% cell viability was lost during the first 24 h of high-gravity SSCF. No beneficial effects of adding selected nutrients were observed in shake flask SSCF. Ethanol concentrations greater than 50 g L−1 led to significant loss of viability and prevented further fermentation in SSCF. The benefits of feeding preadapted yeast cells were marginal at later stages of SSCF. Yeast flocculation did not improve the viability but simplified cell harvest and improved the feasibility of the cell feeding strategy in demo scale. Cultivation at 30 °C instead of 35 °C increased cell survival significantly on solid media containing ethanol and inhibitors. Similarly, in multifeed SSCF, cells maintained the viability and fermentation capacity when the temperature was reduced from 35 to 30 °C during the process, but hydrolysis yields were compromised. By combining the yeast feeding and temperature change, an ethanol concentra-tion of 65 g L−1, equivalent to 70% of the theoretical yield, was obtained in multifeed SSCF on pretreated wheat straw. In demo scale, the process with flocculating yeast and temperature profile resulted in 5% (w/w) ethanol, equivalent to 53% of the theoretical yield.Conclusions: Multifeed SSCF was further developed by means of a flocculating yeast and a temperature-reduction profile. Ethanol toxicity is intensified in the presence of lignocellulosic inhibitors at temperatures that are beneficial to hydrolysis in high-gravity SSCF. The counteracting effects of temperature on cell viability and hydrolysis call for more tolerant microorganisms, enzyme systems with lower temperature optimum, or full optimization of the multifeed strategy with temperature profile.© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Open AccessBiotechnology for Biofuels*Correspondence:  franzen@chalmers.se †Johan O. Westman and Ruifei Wang contributed equally1 Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, SwedenFull list of author information is available at the end of the articlePage 2 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 BackgroundEthanol produced from lignocellulosic materials by fer-mentation is one of the main alternatives for replacing fossil fuels in light-duty transportation. In recent years, the commercialization of lignocellulosic ethanol has gained momentum because of two factors: the improve-ment of enzymes, including reduced cost and the applica-tion of lytic polysaccharide monooxygenases in enzyme cocktails [1, 2], and the construction of several commer-cial-scale production plants [3, 4]. However, inhibitors formed during thermochemical pretreatment of lignocel-luloses, which hinder the growth and fermentation abil-ity of the microorganisms, present a major challenge [5]. Washing the pretreated slurry removes the inhibitors and leads to rapid fermentation, but also leads to loss of fer-mentable sugars and increased demands for wastewater treatment [6, 7].A number of other strategies to reduce deleterious effects of inhibitors have been studied. For example, in situ detoxification has been demonstrated using sulfur oxyanions to sulfonate inhibitory compounds and ren-der them less reactive [8], and laccase has been used to oxidize and polymerize free phenols [9]. The yeast Sac-charomyces cerevisiae is one of the most-used ferment-ing microorganisms for its efficient glucose utilization and high stress tolerance in general. The robustness of yeast cells has been improved by genetic engineering for enhanced capacities in converting inhibitors and main-taining energy/redox balances [10–13]. Knowledge on the mechanisms governing inhibitor tolerance and cellular detoxification in yeasts and other microorganisms is thus of great importance [14–17]. Desirable traits in inhibitor resistance have also been obtained through mutagen-esis, genome shuffling, and evolutionary engineering [18], where a detailed understanding of the mechanisms determining microbial resistance to individual or mul-tiple stresses is not necessary [19]. Pre-exposure of cells to inhibitors during cultivation has been shown to effec-tively improve fermentation performance [20, 21]. The intrinsic detoxification capability of yeast cells has been exploited by using large amounts of inocula [22, 23], increasing the local cell density by encapsulation [24] or flocculation [25], and by continuous fermentation with cell retention [26]. Furthermore, proper implementation of rapidly sedimenting flocculating cells in bioprocesses gives the possibility to omit energy-intensive centrifuga-tion steps [27].Fed-batch simultaneous saccharification and co-fer-mentation (SSCF) can minimize the effects of inhibitors on the process, as the rate at which the inhibitors are introduced can be controlled [10]. In fed-batch SSCF, pretreated raw material is fed to the fermenter where it is continuously hydrolyzed and the released sugars are simultaneously fermented into ethanol. Substrate feed-ing is a practical solution in processes with high solid substrate loadings, also called high-gravity fermentation [28], and promotes xylose and glucose co-consumption [29]. In contrast to other processes for handling high substrate loadings, such as separate hydrolysis and co-fermentation and combined pre-hydrolysis and SSCF, large amounts of glucose are not accumulated in fed-batch SSCF due to the continuous removal of sugars by fermentation. A high xylose-to-glucose ratio can thus be maintained throughout the process, facilitating xylose utilization by recombinant xylose-consuming yeasts by relieving the competitive inhibition by glucose on the xylose uptake via the common glucose/xylose transport-ers [30]. Further development of fed-batch SSCF has been reported toward multifeed SSCF, in which feedings of substrate, enzymes and cells into the SSCF reactor are coordinated in order to balance the main reactions in the process, i.e., to maintain high and balanced rates of both hydrolysis and fermentation [31, 32].Loss of cell viability has been reported in several stud-ies on multifeed, high-gravity SSCF of different substrates [7, 31–33]. The lack of viable cells seems to be the main reason for the declining ethanol production toward the end of the process, which may lead to incomplete utiliza-tion of the available sugars and low overall ethanol yield. Possible reasons for the reduction in viability could be lignocellulose-derived inhibitors [34], lack of nutrients/nitrogen sources [7, 35], lack of unsaturated fatty acids and ergosterol for anaerobic conditions [36], problems associated with high-gravity process or viscous media, e.g., limited mass transfer, high osmolality, and slow reg-ulation of pH and temperature [37], and toxicity of the ethanol produced [38].In this study, we investigate the reasons for the decline in viable cell concentrations in high-gravity SSCF of pretreated wheat straw. It has been reported that yeast flocculation leads to improved tolerance to lignocellu-lose-derived inhibitors [25] and ethanol [39]. The effects of yeast flocculation, to improve cell robustness in gen-eral, and also to improve the feasibility of cell feeding Keywords: Multifeed simultaneous saccharification and co-fermentation (SSCF), High gravity, Yeast viability, Ethanol inhibition, Temperature effect, Combined stress, Flocculation, Demonstration scale, Wheat straw, ModelingPage 3 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 during multifeed SSCF, were investigated. The knowl-edge obtained from these developments of the multifeed SSCF process will be valuable in the further development of flexible and robust lignocellulose-based processes and cell factories, with the objectives to increase the final product concentration and promote complete conversion of the carbohydrates in the lignocellulosic raw material.MethodsStrainsThe metabolically and evolutionarily engineered xylose-fermenting S. cerevisiae strains KE6-12.A (originating from the hybrid diploid wine yeast USM21 via TMB3400) [Albers et  al. unpublished, 40, 41, 42] and IBB10B05 (originating from CEN.PK 113-5D) [43, 44] were used in this study. Both strains were transformed with a gene cassette containing the chimeric flocculation gene FLOw (GenBank accession number KT264162) [45]. The cas-sette was amplified from the genomic DNA of CEN.PK Flow (originating from S. cerevisiae CEN.PK 113-7D (MATa, MAL2-8C, SUC2) [46] with the forward primer 5´-CAGAAAGGGTTCGCAAGTC-3´ and reverse primer 5´-GGCGTATTTCTACTCCAGCATTC-3´ [45]. The resulting PCR product, with flanking regions homol-ogous to the HO locus, was used for homologous recom-bination in the two parental strains mentioned above using the lithium acetate based transformation method [47]. Transformants were selected on yeast extract, pep-tone, dextrose (YPD) plates containing 10  g  L−1 yeast extract (Difco™, BD Biosciences, San Jose, CA), 20 g L−1 peptone (Bacto™, BD Biosciences), 20  g  L−1 glucose (d-glucose monohydrate, Merck KGaA, Darmstadt, Germany), 20  g  L−1 agar (Merck KGaA) and 200  µg/mL G418 (Sigma-Aldrich, Steinheim, Germany). Cor-rect integration into the HO locus (which in itself should not affect cell growth [48]) and the size of the gene were confirmed by PCR using the forward primer 5´-ATGA-TATCCAGTTCGAGTTTATCATTATC-3´ and the reverse primer 5´-CAAATCAGTGCCGGTAACG-3´. The resulting flocculating strains were named KE-Flow and B-Flow.Raw materialsPretreated wheat straw and molasses used in this study were provided by SP Biorefinery Demo Plant (Örnskölds-vik, Sweden). The wheat straw was steam pretreated in a one-step vertical continuous reactor with the addition of 0.2% (w/w)  H2SO4, at pH 2.1–2.2 and 187–188 °C for 6–7 min, by using 11.5 bar steam. After pretreatment, the biomass slurry was separated into a solid fraction and a liquid fraction (pretreatment liquor) using a filter press. The water-insoluble solids (WIS) content in the solid fraction was determined by washing a weighed amount of the moist solid fraction with excess deionized water (the wash was repeated until the glucose remaining in the wash liquid was less than 0.05 g L−1), before drying in an oven at 105 °C for 24 h and weighing of the residual dried material [49]. The solid fraction, in its moist state, was used for hydrolysis and SSCF experiments. The pH of the pretreament liquor was adjusted to 5 using 50% (w/w) NaOH, and it was then filtered by vacuum filtra-tion through sterile disposable bottle top filters with 0.2 µm PES membrane (catalog number 597–4520, Ther-moFisher Scientific, Waltham, MA, USA). The filtered liquor was used in yeast propagation, spotting assays, and the SSCF experiments. The sugar composition in the solid fraction was determined using the National Renew-able Energy Laboratory (NREL) analytical procedure [50].Two batches of pretreated wheat straw, as described previously [32], were used for experiments in shake flasks and 3.6 L Labfors (INFORS HT, Switzerland) bioreactors in this study, and a third batch was used in experiments at demonstration scale and in a 30 L Techfors (INFORS HT, Switzerland) laboratory bioreactor. Material must be produced before each demonstration-scale experiment as the quantity required was too large for long-term storage. The three batches were denoted M1, M2, and M3, and their solid and liquid phases had different compositions (Table 1).Aerobic batch and fed‑batch seed cultivationThe optimal composition of growth media was evalu-ated separately for batch and fed-batch phases; the batch medium in shake flasks at 35 °C and 200 rpm, and the fed-batch medium in bioreactors. The batch medium con-tained molasses as the main carbon source supply, and pretreatment liquor to adapt the cells to the toxic envi-ronment of SSCF. The medium also contained 7.5 g L−1  (NH4)2SO4, 3.5  g  L−1  KH2PO4, 0.7  g  L−1  MgSO4·7H2O, 2 mL L−1 trace metal (TM) solution and 1 mL L−1 vita-min solution. The TM and vitamin solutions were prepared as described previously [51]. Medium composi-tions were investigated at combinations of 5, 7.5, and 10% (v/v) molasses, and 20, 25, and 30% pretreatment liquor. The combination that gave the best compromise between high biomass and low ethanol yield during the 24-h aero-bic cultivation, showing complete utilization of the sug-ars, was selected for batch propagation. The fed-batch medium contained salts as in the batch medium, but no addition of vitamin or TM solutions. The fed-batch phase was started after 24 h of aerobic batch culture. Molasses contents of 0, 2.5, and 5% were investigated in the feed medium, together with 80% pretreatment liquor. The minimal molasses content that produced enough cells for SSCF was selected for feed medium.Page 4 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 Cells from a glycerol stock kept at −80 °C were streaked out on a YPD plate and incubated at 30  °C for approxi-mately 2  days. The liquid inoculum culture was inocu-lated with a single colony from the plate into 50–100 mL batch medium in 250–500 mL Erlenmeyer flasks and was incubated at 30  °C and a shaker speed of 200  rpm for approximately 24 h, after which the cells were harvested by centrifugation at 3000g for 3  min at 4  °C. Flocculat-ing cells were deflocculated by resuspension in 50  mM EDTA followed by washing with 0.9% NaCl solution and final resuspension in 0.9% NaCl. The batch phase of the cell propagation was inoculated to an optical density  (OD600) of 0.5 by addition of the required amount of cell suspension.The yeast cells used in SSCF were propagated in aero-bic batch (0.5 L working volume in 3.6 L Labfors biore-actor, INFORS HT, Switzerland), followed by fed-batch culture, at a dilution rate of 0.05  h−1, sparged with air, with aeration set to 1 vvm, 800  rpm stirring, a temper-ature of 35  °C and pH set to 5.0 by the addition of 3 M NaOH. The working volume was increased to about 2 L in the fed-batch phase and decreased to 0.5 L by harvest-ing part of the culture.At every cell addition to the SSCF reactors, cells were harvested from the propagation reactor. For non-floc-culating cells,  OD600 of the culture was measured, and a separately determined correlation between the OD and the dry cell mass concentration was used to determine how much culture should be harvested. Centrifugation was performed at 3000g for 3  min at 4  °C, and the cell pellet was re-suspended and added to the SSCF reactors. Flocculating cells were allowed to sediment in a stand-ing bottle. The supernatant was poured off and a con-centrated cell suspension was obtained. An aliquot of the cell suspension (about 10 mL) was centrifuged at 3000g for 3  min at 4  °C. A separately determined correlation between the pellet volume and the dry cell weight [cell dry weight (g) = pellet volume (mL)*0.27; R2 of the linear regression was 0.99] was used to determine the cell con-centration in the suspension, and thus how much suspen-sion should be added to the SSCF reactors.SSCF in shake flasksThe Cellic Ctec 2 enzyme preparation (Novozymes, Denmark) was used in all SSCF experiments. The cel-lulase activity of the enzyme preparation was 150  ±  7 FPU  mL−1, measured according to the NREL protocol TP-510-42,628 [52]. Shake flask batch SSCF was carried out with working weight of 100 g in 250 mL baffled Erlen-meyer flasks as described previously [32]. Briefly, the solid fraction of pretreated wheat straw was added to the WIS content that is specified under the corresponding figures. The pH was initially adjusted to 5.0. The medium was supplemented with 0.5  g  kg−1  (NH4)2HPO4. When using 20% WIS, the mixture of solids and enzymes, at 10 FPU g  WIS−1, was preincubated at 50 °C for 2 h prior to cell inoculation, to allow partial liquefaction and mix-ing of the medium. SSCF was initiated by the addition of yeast cells at a cell loading of 0.02  g cell g  WIS−1, after reducing the temperature to 35 °C. This cell loading has previously been shown to be sufficient for efficient fer-mentation in shake flask SSCF on 20% WIS [32]. The flasks were shaken at 180  rpm with no pH control dur-ing fermentation. Samples were taken every 24 h for cell viability and HPLC analysis.SSCF in bioreactorsMultifeed SSCF was performed at working weight around 1  kg in 3.6 L Labfors with one pitched blade impeller and two Rushton impellers, and in 30 L Techfors Table 1 Compositions of solid and liquid fractions for three batches of pretreated wheat strawAll data are average of at least two separate measurements. Data shown are means ± range. M1, M2 and M3 stand for material 1, 2 and 3 received from the SP biorefinery demo plant. The ranges for acetic acid, furfural, and HMF are within 5% of the averagea Data from [32]b Monomeric and oligomeric sugarsSolid fraction (% WIS) Liquid fraction (g L−1)M1a M2a M3 M1a M2 M3Glucan 47.7 ± 3.5 42.4 ± 1.3 36.2 ± 5.0 Glucose 6.8 2.6 ± 0.1b 4.5 ± 0.2bXylan 2.3 ± 0.4 2.6 ± 0.1 4.3 ± 0.1 Xylose 12.8 22.8 ± 1.1b 32.7 ± 1.0bMannan 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.08 Mannose 0.4 0.5 ± 0.1b 1.1 ± 0.1bGalactan 0.04 ± 0.01 0 0.01 ± 0.00 Galactose 1.0 1.0 ± 0.2b 1.9 ± 0.1bArabinan 0.1 ± 0.01 0.08 ± 0.01 0.2 ± 0.00 Arabinose 2.0 2.8 ± 0.03b 4.2 ± 0.1bLignin 41.7 35.6 Acetic acid 3.8 3.2 3.7 ± 0.05Furfural 4.0 0.8 1.5 ± 0.04Total 87.0 76.5 HMF 1.4 0.4 0.3 ± 0.00Page 5 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 bioreactors with two Rushton impellers (INFORS HT, Switzerland). The solid fraction of pretreated wheat straw and yeast cells from a separate propagation reactor were added pulse-wise during the process. All the enzymes were added at the beginning of the process to a concen-tration of 10 FPU g−1 overall WIS, as this has previously been shown to be a suitable process option for wheat straw [32]. The medium was supplemented with 0.5  g  (NH4)2HPO4 and 125 µL Vitahop (a hop-derived fermen-tation enhancer, BetaTech GmbH, Schwabach, Germany) per total kg of working weight, added to reduce the risk of contamination. The reactors were operated without gas sparging.All the experiments were started at an initial WIS of 7%. Solids were added according to predetermined hydroly-sis kinetics [32]. Detailed feed profiles can be found in Additional file  1: Table S1. In the faster feeding profile, feeding was initiated after hydrolysis of 50%, rather than 60%, of the cellulose, according to model predictions, and material was fed to temporarily reach 14% (w/w) calcu-lated apparent WIS in the reactor, rather than 13% (w/w). Samples were collected for the analysis of sugars, fermen-tation products, and residual inhibitors. Multifeed SSCF in bioreactors was carried out at 35 °C, however, in some experiments the temperature was reduced to 30 °C after 24 or 48 h. The pH was controlled at 5.0 by the addition of 3 M NaOH, and the agitation rate was 400 rpm.Determination of cell concentration and viabilityThe cell concentration during aerobic propagation was determined by cell dry weight (CDW) and  OD600. Upon harvest, the flocculating yeast strains KE-Flow and B-Flow were deflocculated with 50  mM EDTA and washed with 0.9% (w/v) NaCl solution before measure-ment. The CDW was measured by first filtering the cell solution through a preweighed 0.45  µm filter (Sarto-rius, Göttingen, Germany), drying for 15 min at 150 W in a microwave oven, and cooling in a desiccator before weighing. The viable cell concentration was determined as colony forming units (CFU) during both cell propaga-tion and SSCF. CFU were determined by plating 0.1 mL of sample, after serial dilution with 0.9% (w/v) NaCl, on YPD plates. The plates were incubated for approximately 2 days at 30 °C before colony counting. The total cell con-centration was determined by counting cells under a light microscope (Leica DM 2000) in a Neubauer improved hemocytometer (Assistent, Glaswarenfabrik Karl Hecht, Germany).Spotting assaysConcentrated (2.5×) YPD agar medium was mixed with filtered pretreatment liquor, ethanol, or both to prepare plates of 1 × YPD agar medium containing in addition to YPD agar, 50% (v/v) liquor, or 50 g L−1 ethanol or both. Upon harvest, the KE-Flow and B-Flow cells were treated with 50 mM EDTA, resulting in complete deflocculation, and washed with 0.9% (w/v) NaCl solution. The defloc-culated cells were re-suspended to an  OD600 of approxi-mately 0.05. Twofold serial dilutions were made from the cell suspension, and 50  μL per dilution was spotted onto the plates. The plates were incubated at 30 or 35 °C for 2  days, after which they were examined and photo-graphed using a Gel Doc scanner (Bio-Rad, Hercules, CA).Analysis of sugars, fermentation products, and inhibitorsFermentation samples were filtered through 0.2-µm nylon filters (VWR, Radnor, PA) prior to storage at −20  °C until analysis. The concentrations of the sugars (glucose, xylose, arabinose, galactose, and mannose) were analyzed using high-performance anion exchange chro-matography on an ICS 3000 system (Dionex, Thermo Scientific, Sunnyvale, CA) with guard and analytical Car-boPac (PA1) columns (Thermo Scientific) maintained at 30  °C, and by electrochemical detection. Milli-Q water was used for sample elution at a flow rate of 1 mL min−1, and 300  mM NaOH was added post-column at a flow rate of 0.5 mL min−1 before the detector. The column was regenerated between sample injections using a mixed eluent consisting of (by volume) 20% Milli-Q water, 40% 300  mM NaOH, and 40% 100  mM NaOH  +  85  mM sodium acetate, followed by equilibration with Milli-Q water. The fermentation products (glycerol, xylitol, and ethanol) and inhibitors [acetic acid, furfural, and 5-(hydroxymethyl)furfural] were analyzed using high-performance liquid chromatography on a Dionex Ulti-mate 3000 system (Dionex, Thermo Scientific) equipped with a Phenomenex Rezex ROA column (Phenomenex, Torrance, CA) and a refractive index (RI) detector (Sho-dex, SHOWA DENKO K.K., Tokyo, Japan). Samples were eluted at 80  °C using 5  mM  H2SO4 at flow rate of 0.8 mL min−1. The concentrations were calculated from calibration curves for standard solutions.Results and discussionEthanol is a major inhibitor in high‑gravity lignocellulosic ethanol productionIn the fermentation of pretreated wheat straw by S. cer-evisiae KE6-12.A in the multifeed SSCF process, the con-centration of viable cells (measured by CFU) decreased to almost zero during the process, regardless of whether more or less toxic pretreated wheat straw were used, and regardless of the scales of the process (Fig.  1a, b). Similar observations have been reported with various raw materials (wheat straw, birch, and spruce) [31–33]. These results indicated that the fermentation conditions Page 6 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 in these lignocellulosic media were not suitable for sus-taining cell viability. Cell feeding, instead of adding all the cells at the beginning of fermentation, allowed for a more effective use of the same total amount of cells, and the fermentation capacity was maintained for a longer period. However, cell feeding was not sufficient to stop the decline in the viable cell population or to ensure com-plete fermentation of the released sugars at the end of the process (Fig. 1b).To investigate whether the observed decrease in via-bility during SSCF was due to some nutrient limitation, yeast extract (2  g  kg−1), vitamin solution (1  mL  kg−1), trace metal (TM) solution (2  mL  kg−1) [51], biotin (1  mg  kg−1), or inorganic salts [7.5  g  kg−1  (NH4)2SO4, 3.5  g  kg−1  KH2PO4 and 0.7  g  kg−1  MgSO4·7H2O] were added to shake flask SSCF at a low content of water insoluble solids (WIS, 7% w/w). None of them led to sig-nificantly improved ethanol production or an increase in cell viability (Fig.  1c) The addition of nutrients was fur-ther tested in 20% WIS shake flask SSCF, in which the inhibitor concentrations were much higher. The same amounts of vitamin and TM solutions, biotin, and inor-ganic salts as above were used. In addition, the anaerobic growth factor ergosterol (10  mg  kg−1) and Tween 80 (420  mg  kg−1) were added, but no improvements in ethanol production were observed, and the cell viability decreased to zero within 48 h in all cases (Fig. 1d). The medium composition in control experiments was the same as that in multifeed SSCF. It was therefore con-cluded that the absence of viable cells at later stages of multifeed SSCF was not due to nutrient limitation.Other factors causing stress during multifeed SSCF are the inhibitors present in the raw material and the ethanol produced. However, inhibitor concentrations do not increase to high levels in a fed-batch process due to dilution effects and continuous conversion by the yeast. In fact, a decrease in aromatic aldehyde concentrations and an almost constant acetic acid concentration were observed (data not shown). The only potentially toxic compound that increased significantly in concentration over time was the fermentation product, ethanol. Etha-nol inhibition has so far not been seen as a major prob-lem in lignocellulosic ethanol production, and has only been mentioned as a potential issue in high-gravity fer-mentation [37]. It is only recently that the ethanol titers reported from lignocellulosic feedstocks have exceeded Fig. 1 Decrease of cell viability during multifeed SSCF and limited effects of nutrient supplementation. Cell viability (% of CFU/total cell counts) and concentrations of glucose and ethanol during a laboratory-scale SSCF of material M1 with all the yeast added initially; and b demonstration-scale SSCF with feeding of yeast and substrate material M2. Ethanol concentration and cell viability during c 7% WIS and d 20% WIS (with 2-h pre-hydrolysis) shake flask SSCF of material M1 with additions of indicated nutrients. S. cerevisiae KE6-12.A was used, and the temperature was 35 °C in all cases. Arrows in (a–b) indicate the loading of yeast cells. TM trace metals. Error bars show the results of duplicate experiments. Error bars for ethanol concentration in (c–d) are smaller than the symbols. The measured cell viability in (d) was 0 at 48 h for all cases. Panels a and b are adapted from [32]Page 7 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 the benchmark of 4% (w/w). These titers are still far below those achieved with sucrose- and starch-based substrates, of 8–12% (v/v) [53, 54], and lower than the levels generally considered to be lethal to the yeast S. cer-evisiae. For example, the wild-type lab strain of BY4741 has been cultured in the presence of 10 and 12.5% (v/v) ethanol for 30 h, with remaining viabilities of 80 and 30%, respectively [55].To investigate the significance of inhibition by etha-nol, different levels of ethanol were added to 20% (w/w) WIS shake flask SSCF experiments. Without etha-nol addition, approximately 40  g  L−1 ethanol was pro-duced within the first 24 h (i.e., an average fermentation rate of 1.6  g  L−1  h−1), after which the fermentation rate decreased to 0.1  g  L−1  h−1 between 24 and 48  h. When approximately 50 g L−1 ethanol was added, some ethanol production was evident during the first 24  h (~0.5 g L−1 h−1), after which production ceased and the ethanol concentration decreased, probably due to evapo-ration (−0.04 g  L−1 h−1 from 24 to 48 h). The amount of ethanol produced was only 25% of that produced in the control case. When 75  g  L−1 ethanol was added at the start of fermentation, virtually no ethanol production was seen in the shake flasks (0.08  g  L−1  h−1 from 0 to 24 h) (Fig. 2). These results clearly show that at concen-trations of about 50 g L−1 and above, ethanol is already a major inhibitor of fermentation in SSCF with 20% WIS of pretreated wheat straw. A concentration range of 40–60 g L−1 of ethanol has been observed to be a turning point where drastic reduction in growth rate and prolon-gation of the lag-phase occur for several laboratory and industrial S. cerevisiae strains cultivated in the medium of spruce hydrolysate [56].To investigate whether ethanol also inhibits the hydro-lytic enzymes, enzymatic hydrolysis was carried out in shake flasks on 20% WIS with the addition of 75  g  L−1 ethanol. The results showed a 12% decrease in glucose release (Additional file 1: Figure S1). Therefore, inhibition of the enzymes by ethanol was not the main reason for the observed decreased ethanol production in the SSCF experiments with ethanol supplementations.From these results, it is clear that at concentrations of 40–50  g  L−1, ethanol became inhibitory to S. cer-evisiae KE6-12.A in SSCF at 20% WIS, with 0.02 g DW (g WIS)−1 inoculation. Our results indicate that in high-gravity lignocellulosic ethanol production, ethanol inhi-bition has to be alleviated so that further increases in ethanol titer would be possible.Flocculation simplifies cell feeding in the multifeed SSCF processIt has been shown that when cells agglomerate and form dense flocs, their tolerance to ethanol [39] and to furan aldehydes [25] is improved. Thus, flocculation could be a promising strategy to enhance the performance of S. cerevisiae in high-gravity, lignocellulosic ethanol produc-tion, especially to increase the ethanol titer. Furthermore, flocculating cells would rapidly sediment at the bottom of the reactor making cell recovery and processing easier.The flocculating strain KE-Flow formed dense cell aggregates approximately 3–4  mm in size when culti-vated in shake flasks with liquid medium. However, no improvement was observed with the KE-Flow strain compared to that with the KE6-12.A strain in shake flask SSCF (Additional file 1: Figure S2), or multifeed SSCF in bioreactors, regardless of whether all the yeast was added initially or at different times (Fig. 3). Fermentation was so slow in the later stages of SSCF that the released glucose accumulated in the reactor, and the viability decreased despite the flocculation. This suggests that dense cell flocs were not successfully formed in the SSCF reac-tor [25]. The SSCF medium contained a high amount of WIS, i.e., undissolved particles. These particles, together with the mixing in the reactor, probably disrupted larger flocs. These results also suggest that flocculation might not protect cells from ethanol inhibition under the condi-tions used in SSCF.Although no improvement in performance was observed in SSCF when using flocculating cells, rapid sedimentation of the KE-Flow cells was observed in the propagation reactor (Fig.  4). The strong flocculation is typical for the Flo1 phenotype [57], which is illustrated by these mutants. The strain may thus be attractive from a process point of view. Using flocculating cells may Fig. 2 Ethanol inhibition in high-gravity SSCF with S. cerevisiae KE6-12.A. Shake flask SSCF of 20% (w/w) WIS of steam-pretreated wheat straw (material M1) with 2-h pre-hydrolysis, 10 FPU (g WIS)−1 enzyme dosage, at 35 °C. The addition of approximately 75 or 50 g L−1 ethanol severely inhibited ethanol production. Results shown are average values of duplicate experiments, and the relative difference between duplicate experiments was lower than 5%Page 8 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 Fig. 3 Multifeed SSCF with the non-flocculating strain KE6-12.A and the flocculating strain KE-Flow. Accumulated WIS concent and percent viability (a, b) and concentrations of ethanol, glucose, and xylose (c, d) using: (a, c) KE6-12.A; and (b, d) KE-Flow in multifeed SSCF at 22% (w/w) overall WIS of material M1, at 35 °C. Cells were added initially (w/o cell feed, filled symbols) or fed during the process (w cell feed, open symbols). Feeding of cells is indicated by the arrows. The detailed feeding profile has been described elsewhere [32]. Values are averages from duplicate experiments, and error bars show the results from the individual experimentsFig. 4 Sedimentation of KE-Flow cells in propagation reactor. At t = 0 s, stirring and aeration were completely stopped. Pretreatment liquor from material M2 was used in this casePage 9 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 simplify the harvesting and feeding of cells to the SSCF reactor.High temperature exacerbates inhibition by ethanol and pretreatment liquorAs discussed above, inhibition by ethanol in high-gravity SSCF was greater than that expected for S. cerevisiae. It is unlikely that ethanol at a concentration of 40–50 g L−1 was solely responsible. Rather, the inhibition was due to the combined effect of ethanol, lignocellulose-derived inhibitors, and stressful SSCF conditions. In the SSCF or, generally, simultaneous saccharification and fermen-tation (SSF) setups, the temperature is a compromise between the optimal temperatures for the hydrolytic enzymes (45–50  °C for Cellic CTec2, Novozymes) and the fermenting microorganism (around 30  °C for S. cer-evisiae) [58]. This may cause additional stress on the cells in the multifeed SSCF. Xiros and Olsson showed that using preadapted yeast cells and yeast extract improved the ethanol yield about 30 times at 30 °C in SSF of spruce at 20% WIS, but at higher temperatures, the effectivity of this strategy decreased [7]. These findings led us to hypothesize that the ethanol produced, in combination with the inhibitors and the relatively high temperature for the yeast (35 °C) caused the decrease in viability and fermentation capacity.To investigate the effects of combined stresses on yeast growth, spotting assays were performed at 35 and 30 °C. The results clearly showed that incubation at the higher temperature strongly increased the inhibitory effects of both ethanol and the pretreatment liquor (Fig.  5). With the combination of ethanol and pretreatment liquor, almost no cells grew at 35 °C, showing a strong additive, possibly synergistic, effect of the three factors resembling conditions in high-gravity SSCF. This is probably the reason why no fermentation was observed when a high concentration of ethanol was added to SSCF (Fig. 2), and why further fermentation was difficult with ethanol con-centrations greater than 50 g L−1, in spite of adding fresh cells during the process.Similar results were observed in spotting assays with cells obtained at different time points during the fed-batch cell propagation (data not shown). Ethanol and elevated temperature could affect both the fluidity and structure of the cell membrane [59, 60], making the cells more permeable [61] and thus vulnerable when toxic compounds are present. Similar results were also observed when the xylose-fermenting, evolutionary engi-neered S. cerevisiae strain IBB10B05 [43] was used (Addi-tional file  1: Figure S3), suggesting that the combined inhibition due to ethanol, inhibitors, and temperature is not strain specific.The YPD plates were incubated aerobically, indicating that the inability of the yeast to survive the combined stress was not due to a lack of sterols or oleic acid, since S. cerevisiae can synthesize these under aerobic con-ditions. This is consistent with the results of the ergos-terol-supplemented batch SSCF experiments (Fig. 1d), in which the addition of sterols and oleic acid (Tween 80) did not improve the ethanol production.Fig. 5 Spotting assay of the KE-Flow cells. YPD agar plates were supplemented with 50 g L−1 ethanol, or 50% (v/v) pretreatment liquor (from material M2), or both. The cells were harvested after 24 h of fed-batch propagation. The plates were incubated at 35 or 30 °C for 48 h. Duplicate cell dilution and plating showed similar resultsPage 10 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 Reducing the temperature during SSCF improves cell viabilityIn order to achieve sustained fermentation and complete sugar utilization, the inhibition caused by ethanol, inhibi-tors, and the process temperature must be alleviated. Decreasing the temperature during SSCF could lead to improved cell viability and thus more complete fermenta-tion. However, the risk of less complete hydrolysis has to be considered.The temperature in the multifeed SSCF was reduced from 35 to 30  °C after 24  h. At this point, the ethanol concentration had reached approximately 40–50  g  L−1 (cf. Fig. 3). The reduction in temperature, together with cell feeding, led to increased viability and sustained fermentation capacity until the end of the process, with no accumulation of glucose (Fig. 6). In the 30 °C case, an increase in the fermentation rate was observed when a large amount of substrate was added at 120 h. In a similar process carried out at a constant temperature of 35  °C, glucose accumulated after the same substrate addition, indicating a decrease in fermentation, and a 26% lower ethanol yield on total sugars was measured at 168 h. The measured viable cells at the later stages were significantly lower in the process carried out isothermally at 35  °C, compared to the process with temperature reduction. The decrease in fermentation and accumulation of glu-cose started already between 96 and 120  h, indicating that the number of viable cells might be insufficient for taking up all glucose released via enzymatic hydrolysis (Fig.  6). Reducing the temperature likely decreases the rate of cellulose hydrolysis leading to lower glucose con-centrations; however, very similar ethanol profiles were obtained for both cases before 120 h. This seems due to the enhanced xylose utilization observed in the process with a decrease in the temperature (Fig. 6). The glycerol formation followed the ethanol production (see Addi-tional file  1: Figure S4, where also acetate, furfural, and HMF concentrations are shown).In a previous study, Mutturi and Lidén instead applied a linear increase in temperatures ranging from 32  °C to 45 °C in SSF of pretreated spruce and Arundo [62]. When using Arundo, a higher ethanol titer was obtained in SSF with the temperature profile, than those in processes operated at a constant temperature of either 32 or 39 °C. This was likely due to the fact that, first, the increasing temperature would promote enzymatic hydrolysis com-pared with a constant temperature at 32 °C; and second, a gradual increase in temperature may give cells better chances to adapt than when using a constant high tem-perature from the beginning [62]. However, it is doubtful whether this strategy would work with more toxic sub-strates and at higher ethanol concentrations. In the same study, when pretreated spruce which contained higher amount of inhibitors was used, no difference in ethanol titers was observed between the processes with the tem-perature profile and with a constant temperature 39  °C (32.2 and 33.1  g  L−1, respectively) [62]. These ethanol concentrations were considerably lower than the ones obtained herein, which may explain the different observa-tions since they were below the level where we observed significant inhibition.In conclusion, a temperature profile with a decrease in temperature after a certain period, combined with cell feeding, may offer a suitable process strategy for sus-tained viability and fermentation capacity at high ethanol titers.Fig. 6 Multifeed SSCF at constant temperature or with temperature reduction. a Cell viability (% of CFU/total cell counts) and accumu-lated WIS content (indicating substrate feeding); and b concentration of glucose, xylose, and ethanol in 25% (w/w) WIS multifeed SSCF of material M2, with the KE-Flow strain, at a constant temperature of 35 °C, or at 35 °C for 24 h, and then at 30 °C. Arrows between the panels indicate cell additions. Details of the substrate and cell feeding can be found in Additional file 1: Table S1Page 11 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 Adapting multifeed SSCF to KE‑Flow and a new material batch increased ethanol productionAs shown previously, the flocculating yeast strain KE-Flow performed similar to the parental KE6-12.A strain when it was propagated and used in fermentation pro-cesses that had been designed for KE6-12.A and material M1 (Fig. 3, Additional file 1: Figure S2; Table S2) [32]. As the material M2 had different compositions from that of M1 (Table 1), the media used in the propagation step and the feeding profile in the multifeed SSCF process were adjusted for the KE-Flow strain and material M2.In the propagation, the objective was to maximize the use of the pretreatment liquor and minimize the use of molasses, while generating enough cells with consistent capacity for fermentation in SSCF. The medium contain-ing 5% (v/v) molasses and 25% (v/v) pretreatment liquor was selected for the initial batch propagation, and the medium with 2.5% molasses and 80% pretreatment liq-uor was selected for fed-batch cultivation (for details see Additional file 1: Figure S5).The objectives of re-designing the multifeed SSCF were to speed up the substrate feeding and to maximize the use of pretreatment liquor. Faster solids feeding, while avoiding mixing problems, is preferable because it pro-longs the average time that substrates are available for hydrolysis. In a process with temperature reduction, a larger fraction of substrates would be loaded at the higher temperature, which may improve the overall hydrolysis.The faster substrate feeding was developed by using the open loop approach described previously [32], with adjustments in the threshold values used to calculate when and how much substrate should be added. In brief, the rate of hydrolysis of the substrate was predicted using a kinetic model. When the predicted substrate conver-sion reached a lower threshold, 50% instead of the 60% that was used in the previous experiments, a feeding event was triggered. The amount of feed was calculated from the current concentration of WIS and the maxi-mum concentration of WIS that could be adequately mixed in the reactor. For material M2, the lab reactors could mix medium containing up to 14% (w/w) WIS, while for material M1 the upper limit was 13% (w/w). The reason for the difference in the two material batches was probably that a sieve was used to homogenize the size of the solid clumps of M2 material at the demo plant. Start-ing feeds earlier and adding more substrates every time resulted in a faster substrate feeding profile (Additional file 1: Table S1). The new feeding was implemented suc-cessfully in laboratory-scale multifeed SSCF of material M2 without any mixing problems.The use of pretreatment liquor in SSCF instead of water was also investigated together with the new feeding profile. The pretreatment liquor contained sugars, thus contributing to an increase in ethanol production in the early stage of the process (Fig. 7). Ethanol titers of greater than 60  g  L−1 were obtained at 96  h, and 65  g  L−1 was reached after 144 h of SSCF at 22% overall WIS, equiva-lent to 70% of the theoretical ethanol yield from the total sugar inputs. This represents a total process ethanol yield of 186  L/ton dry straw. The calculated yield at 120  h, which can be considered a more realistic process time, is given in Additional file 1: Table S2. The relatively low overall yields reflect the facts that some of the pretreated material is used to replace other sugars during cell propa-gation, and that both hydrolysis and fermentation were incomplete.Fig. 7 Adapted multifeed SSCF with faster feeding and maximum use of pretreatment liquor. a Cell viability (% of CFU/total cell counts) and accumulated WIS content (indicating substrate feeding); and b concentration of glucose, xylose, and ethanol in 22% (w/w) WIS multifeed SSCF of material M2, with the KE-Flow strain, at a constant temperature of 35 °C, or at 35 °C for 24 h, and then at 30 °C. Arrows between the panels indicate cell additions. Values are averages from duplicate experiments, and the error bars show the results of the individual experiments. Details of the substrate and cell feeding can be found in Additional file 1: Table S1Page 12 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 Decreasing the process temperature after 24  h improved the viability of the cells and the consumption of xylose and available glucose (Fig.  7). The measured glucose concentration was close to zero at the lower temperature. Between 24 and 72 h, less ethanol was pro-duced than at the higher temperature. Therefore, less glucose must have been available for fermentation at the lower temperature, which means the hydrolysis rate was lower. The limited hydrolysis was also illustrated by the higher residual WIS and the proportion of glucose in the WIS at the end of the process, indicating a larger residual amount of unhydrolyzed cellulose at the lower tempera-ture (Table 2). Glycerol was formed at higher concentra-tions at the lower temperature, which is compatible with improved growth and/or higher xylose conversion (Addi-tional file 1: Figure S6). The high ethanol concentrations obtained until 96 h during isothermal operation at 35 °C illustrates the benefit of feeding cells during the process (Fig.  7). Further optimization is necessary for balancing the negative effect of a lower temperature on hydrolysis vs. the beneficial effects on ethanol tolerance, viability, and fermentative activity.Scale‑up: opportunities and challenges when using flocculating yeast and a temperature profileTo investigate the feasibility of using flocculating yeast in an industrial setting, the re-designed yeast propagation and multifeed SSCF processes (Additional file  1: Table S1) were carried out in 10 m3 reactors at the SP Biorefin-ery Demo Plant in Örnsköldsvik, Sweden. A new batch of material (M3) was produced. The furfural and xylose concentrations in the pretreatment liquor from this batch were higher than those in the M2 material used during the optimization of the laboratory-scale processes (the detailed compositions are given in Table 1).When cultivated aerobically in the demonstration scale (10  m3) on the new material, the growth of the KE-Flow cells was similar to that in the laboratory during the batch phase and the fed-batch phase until the first cell withdrawal (Additional file 1: Figure S7). At the first harvest, cells were concentrated by sedimentation inside the propagation reactor, because agitation and aeration were stopped. A high cell density slurry  (OD600 of ~200) was thus obtained by harvesting the cells from the bottom of the reactor. The cell slurry was used directly as inoculum for SSCF.After the first harvest, the cells did not re-suspend prop-erly upon restarting stirring and aeration. Cell growth was therefore insufficient for the planned subsequent cell feedings to the SSCF reactor (Additional file 1: Figure S7). Hence, the cell feeding that was planned at 12 h was omit-ted, and the corresponding amount of cells was instead added with the last two feedings (at 30 and 52 h). Using a sedimentation tank next to the propagation reactor, as was done at the laboratory scale, would have improved the pro-cess by reducing the disturbance of the propagation process.Since the hydrolysis rate was lower at the lower tempera-ture of 30  °C (Table  2), further optimization of substrate feeding and temperature profile was required. The initial period of higher temperature was extended to 48 h in order to improve hydrolysis, and a final cell feeding was per-formed at 52 h. Actual cell dry weights added in three cell feedings were about 13, 1.3 and 3 kg, respectively. Before the last cell addition, the percentage of viable cells was 3%. After the temperature reduction, the decrease in viable cells was about 85% slower than during the 23 h (Fig. 8a) before the temperature reduction. Furthermore, the etha-nol production rate appeared to be constant until the end of the process (Fig. 8c). In the end, the percentage of viable cells over total cells was approximately 8%. Of the cells added at the last feeding, approximately 45% remained via-ble, so the lower temperature clearly improved cell viability in the demonstration scale as well. However, the concen-tration of viable cells was much lower than in the labora-tory-scale experiments at the corresponding time points, also after the decrease in temperature (Figs. 7a, 8a).After 24  h of fermentation, the residual glucose con-centration in the reactor leveled out at approximately 10 g L−1, and the xylose concentration at approximately 25  g  L−1 (Fig.  8c). This indicates that fermentation was significantly slower than on the laboratory scale. The dif-ference in performance was probably due to the problems encountered in handling the flocculating yeast and pro-cess control on the larger scale. It was found, for exam-ple, that the actual WIS content in the demo reactor was higher than planned, meaning that too much solid mate-rial or too little liquid had been added to the reactor. The resulting high viscosity led to reduced mixing, hydroly-sis, and fermentation. Another reason may be differences in the toxicity of the material, as M3 contained signifi-cantly higher amounts of inhibitors than M2 (Table  1). Table 2 Residual WIS (%, w/w) and sugar composition of the WIS [%, i.e., g (100 g WIS)−1] at 120 h of multifeed SSCFThe values given are the average of duplicate experiments ± the span to minimum and maximum valuesTemperature of SSCF WIS Glucose Xylose Mannose d‑Galactose l‑Arabinose35 °C 11.6 ± 0.8 11.9 ± 3.5 1.3 ± 0.2 1.1 ± 0.1 0.1 ± 0.0 0.2 ± 0.035 → 30 °C 13.0 ± 0.1 21.2 ± 0.6 1.7 ± 0.1 1.1 ± 0.0 0.0 ± 0.0 0.2 ± 0.0Page 13 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 The concentration of acetate in the fermentation broth increased to 4.6 g L−1 at 96 h, while the glycerol concen-tration was lower than those in previous experiments, reflecting both poor growth and low xylose consumption (Additional file 1: Figure S8).Although many adjustments and compromises had to be made to operate multifeed SSCF at the demonstra-tion plant, ethanol titers above 5% (w/w) were achieved. Toward the end of the process, small amounts of lac-tic acid were formed, and some bacterial colonies were found on the YPD plates used for CFU determination. However, since the lactate concentration was very low (<0.3  g  L−1) and available glucose in the medium was high, contamination was not a major problem during these experiments.The multifeed SSCF experiment could not be repeated on the demo scale due to limited resources, but was repeated on an intermediate scale in the laboratory using the same material. The demo process was scaled down to a 30 L Techfors reactor, i.e., roughly 10 times the scale of the 3.6 L Infors reactors used for process development. The fermentation results were consistent with those obtained from the demo plant experiment (Fig. 8; Addi-tional file 1: Figure S8). Neither of them reached the high ethanol titers obtained during the laboratory-scale exper-iments using material M2 (Fig.  7). No issues with the mixing in the fermenter were observed during the experi-ment in the Techfors reactor. It could therefore be con-cluded that differences between the materials obtained after pretreatment were the main contributor to the dif-ferent fermentation results.Apart from demonstrating scalability of the process, the results from larger scales highlight the importance of consistency in the pretreatment process, in order to obtain material with similar hydrolyzability and inhibi-tion characteristics. However, this is difficult to achieve due to variations in feedstocks and difficulties in opera-tion on a large scale. Thus, flexibility in the SSCF process is necessary. The multifeed SSCF process is an example of an adaptable process. Adjustments can be made to the Fig. 8 Multifeed SSCF of material M3 in 10 m3 and 30 L reactors with KE-Flow. SSCF was carried out at 35 °C for 48 h and then at 30 °C. a Cell viabil-ity (% of CFU/total cell counts), planned accumulation, and measured residual WIS (%, w/w) during the demonstration-scale experiment, and  b the same experiment on an intermediate scale in a 30 L Techfors reactor; c concentration of sugars and fermentation products during the demo-scale experiment, and d during the  reactor experiment. Arrows between the panels indicate cell additionsPage 14 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 process based on the toxicity of the material by chang-ing the amount of pretreatment liquor in the propaga-tion step and in the SSCF, together with modifications of the feeding of cells and solids. To enable such adjust-ments in real time, rapid sampling methods and online measurements of sugars, inhibitors, and viable cells will be required to provide information on the status of the process, and to make feedback control of multifeed SSCF possible.ConclusionsResults from laboratory- and demonstration-scale experiments showed the feasibility of utilizing flocculat-ing yeast cells as an efficient way to concentrate cells for use in multifeed SSCF. Ethanol inhibits fermentation at a concentration of 50  g  L−1 in the presence of lignocel-lulosic inhibitors. Flocculation did not provide a solu-tion to the ethanol inhibition in SSCF, but is an attractive strategy for facilitating cell harvesting and processing. The fermentation capacity of the cells can be maintained, and higher ethanol titers can be achieved by reducing the temperature in the SSCF reactor when the ethanol con-centration reaches an inhibitory level. These changes, together with faster substrate feeding, and replacing water with pretreatment liquor, led to an ethanol titer of 65 g L−1 with maintained cell viability and almost com-plete utilization of the fermentable sugars. Optimal per-formance of multifeed SSCF requires adaptation of the process to each material used.Authors’ contributionsJW designed the study, constructed the strains TE-Flow and B-Flow, performed experiments, analyzed and interpreted data, and wrote the manuscript. RW conceived and designed the study, performed the mathematical modeling, performed experiments, analyzed and interpreted data, and wrote the manu-script. VN designed the study, performed experiments, interpreted data, and participated in the writing of the manuscript. CJF conceived and designed the study, interpreted data, and wrote the manuscript. All  authors read and approved the final manuscript.Author details1 Division of Industrial Biotechnology, Department of Biology and Biologi-cal Engineering, Chalmers University of Technology, Gothenburg, Sweden. Additional fileAdditional file 1: Figure S1. Ethanol inhibition in high-gravity enzy-matic hydrolysis with enzyme preparation Cellic Ctec 2. Figure S2. Shake flask SSCF with the non-flocculating strain KE6-12.A and the flocculating strain KE-Flow. Figure S3. Spotting assay of the B-Flow cells. Figure S4. Multi-feed SSCF at constant temperature or with temperature reduc-tion. Figure S5. Investigation of medium compositions for batch and fed-batch propagation of KE-Flow with material M2. Figure S6. Adapted multi-feed SSCF with faster feeding and maximum use of pretreatment liquor. Figure S7. Comparison of cultivation of KE-Flow in 3.6 L laboratory reactors and in the demonstration plant. Figure S8. Multi-feed SSCF of material M3 in 10  m3 and 30 L reactors with KE-Flow. Table S1. SSCF feed-ing schemes for laboratory scale reactors. Table S2. Summary of results from multi-feed SSCF experiments using steam pre-treated wheat straw.2 Present Address: Chr. Hansen A/S, Bøge Allé 10-12, 2970 Hørsholm, Denmark. 3 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria. 4 Present Address: Forest Products Biotechnol-ogy, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T1Z4, Canada. AcknowledgementsThe authors are grateful to Prof. Bernd Nidetzky from the Graz University of Technology for providing the strain IBB10B05, and for facilitating VN’s visit at the Chalmers University of Technology.Competing interestsThe authors declare they have no competing interests.Availability of data and materialsData and material will be made available upon request.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.FundingThis study was funded by the Swedish Energy Agency (Grant Number P37353-1) and by the Chalmers Energy Initiative (http://www.chalmers.se/en/areas-of-advance/energy/cei/). The funding bodies had no influence on the design of the study and were not involved in the collection, analysis, and interpretation of data, nor in the writing of the manuscript.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub-lished maps and institutional affiliations.Received: 18 January 2017   Accepted: 24 August 2017References 1. Cannella D, Jørgensen H. Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol produc-tion? Biotechnol Bioeng. 2014;111(1):59–68. 2. Johansen KS. Discovery and industrial applications of lytic polysaccharide mono-oxygenases. Biochem Soc Trans. 2016;44(1):143–9. 3. Bardhan S, Jose S, Godsey L. Cellulosic biofuel in the United States. In: Jose S, Bhaskar T, editors. Biomass and biofuels: advanced biorefineries for sustainable production and distribution. Boca Raton: CRC Press; 2015. p. 301. 4. Guo M, Song W, Buhain J. Bioenergy and biofuels: history, status, and perspective. Renew Sust Energ Rev. 2015;42:712–25. 5. Jönsson LJ, Alriksson B, Nilvebrant N-O. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels. 2013;6:16. 6. Li H, Chen H. Detoxification of steam-exploded corn straw produced by an industrial-scale reactor. Proc Biochem. 2008;43(12):1447–51. 7. Xiros C, Olsson L. Comparison of strategies to overcome the inhibitory effects in high-gravity fermentation of lignocellulosic hydrolysates. Biomass Bioenerg. 2014;65:79–90. 8. Cavka A, Alriksson B, Ahnlund M, Jönsson LJ. Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors. Biotechnol Bioeng. 2011;108(11):2592–9. 9. Jurado M, Prieto A, Martínez-Alcalá Á, Martínez ÁT, Martínez MJ. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour Technol. 2009;100(24):6378–84. 10. Almeida JRM, Modig T, Petersson A, Hahn-Hägerdal B, Lidén G, Gorwa-Grauslund MF. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol. 2007;82(4):340–9.Page 15 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213  11. Ask M, Mapelli V, Höck H, Olsson L, Bettiga M. Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibi-tors in pretreated lignocellulosic materials. Microb Cell Fact. 2013;12:87. 12. Wei N, Quarterman J, Kim SR, Cate JHD, Jin Y-S. Enhanced biofuel produc-tion through coupled acetic acid and xylose consumption by engineered yeast. Nat Commun. 2013;4:2580. 13. Liu ZL. Molecular mechanisms of yeast tolerance and in situ detoxi-fication of lignocellulose hydrolysates. Appl Microbiol Biotechnol. 2011;90(3):809–25. 14. Wang X, Yomano LP, Lee JY, York SW, Zheng H, Mullinnix MT, Shanmugam KT, Ingram LO. Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proc Nat Acad Sci USA. 2013;110(10):4021–6. 15. Koopman F, Wierckx N, de Winde JH, Ruijssenaars HJ. Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degrada-tion pathways of Cupriavidus basilensis HMF14. Proc Nat Acad Sci USA. 2010;107(11):4919–24. 16. Lindahl L, Genheden S, Eriksson LA, Olsson L, Bettiga M. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii. Biotech-nol Bioeng. 2016;113(4):744–53. 17. Gu H, Zhang J, Bao J. High tolerance and physiological mechanism of Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue. Biotechnol Bioeng. 2015;112(9):1770–82. 18. Demeke MM, Dietz H, Li Y, Foulquié-Moreno MR, Mutturi S, Deprez S, Den Abt T, Bonini BM, Liden G, Dumortier F, Verplaetse A, Boles E, Thevelein JM. Development of a D-xylose fermenting and inhibitor tolerant indus-trial Saccharomyces cerevisiae strain with high performance in lignocel-lulose hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels. 2013;6:89. 19. Çakar ZP, Seker UOS, Tamerler C, Sonderegger M, Sauer U. Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae. FEMS Yeast Res. 2005;5(6–7):569–78. 20. Alkasrawi M, Rudolf A, Lidén G, Zacchi G. Influence of strain and cultiva-tion procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce. Enz Microb Technol. 2006;38(1–2):279–86. 21. Nielsen F, Tomás-Pejó E, Olsson L, Wallberg O. Short-term adaptation dur-ing propagation improves the performance of xylose-fermenting Saccha-romyces cerevisiae in simultaneous saccharification and co-fermentation. Biotechnol Biofuels. 2015;8:219. 22. Cantarella M, Cantarella L, Gallifuoco A, Spera A, Alfani F. Comparison of different detoxification methods for steam-exploded poplar wood as a substrate for the bioproduction of ethanol in SHF and SSF. Proc Biochem. 2004;39(11):1533–42. 23. Pienkos PT, Zhang M. Role of pretreatment and conditioning pro-cesses on toxicity of lignocellulosic biomass hydrolysates. Cellulose. 2009;16(4):743–62. 24. Westman JO, Manikondu RB, Franzén CJ, Taherzadeh MJ. Encapsulation-induced stress helps Saccharomyces cerevisiae resist convertible lignocel-lulose derived inhibitors. Int J Mol Sci. 2012;13(9):11881–94. 25. Westman JO, Mapelli V, Taherzadeh MJ, Franzen CJ. Flocculation causes inhibitor tolerance in Saccharomyces cerevisiae for second-generation bioethanol production. Appl Environ Microbiol. 2014;80(22):6908–18. 26. Ylitervo P, Franzén C, Taherzadeh M. Impact of furfural on rapid ethanol production using a membrane bioreactor. Energies. 2013;6(3):1604–17. 27. Westman JO, Franzén CJ. Current progress in high cell density yeast bio-processes for bioethanol production. Biotechnol J. 2015;10(8):1185–95. 28. Hodge DB, Karim MN, Schell DJ, McMillan JD. Model-based fed-batch for high-solids enzymatic cellulose hydrolysis. Appl Biochem Biotechnol. 2009;152(1):88–107. 29. Olofsson K, Rudolf A, Lidén G. Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J Biotechnol. 2008;134(1–2):112–20. 30. Olofsson K, Wiman M, Lidén G. Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co-fermenta-tion for bioethanol production. J Biotechnol. 2010;145(2):168–75. 31. Wang R, Koppram R, Olsson L, Franzén CJ. Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol. Bioresour Technol. 2014;172:303–11. 32. Wang R, Unrean P, Franzén CJ. Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production. Biotechnol Biofuels. 2016;9:88. 33. Koppram R, Olsson L. Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings. Biotechnol Biofuels. 2014;7:54. 34. Palmqvist E, Hähn-Hägerdal B. Fermentation of lignocellulosic hydro-lysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol. 2000;74(1):25–33. 35. Jorgensen H. Effect of nutrients on fermentation of pretreated wheat straw at very high dry matter content by Saccharomyces cerevisiae. Appl Biochem Biotechnol. 2009;153(1–2):44–57. 36. Andreasen AA, Stier TJB. Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Comp Physiol. 1953;41(1):23–36. 37. Koppram R, Tomas-Pejo E, Xiros C, Olsson L. Lignocellulosic ethanol pro-duction at high-gravity: challenges and perspectives. Trends Biotechnol. 2014;32(1):46–53. 38. Stanley D, Bandara A, Fraser S, Chambers PJ, Stanley GA. The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae. J Appl Microbiol. 2010;109(1):13–24. 39. Smukalla S, Caldara M, Pochet N, Beauvais A, Guadagnini S, Yan C, Vinces MD, Jansen A, Prevost MC, Latgé J-P, Fink GR, Foster KR, Verstrepen KJ. FLO1 Is a variable green beard gene that drives biofilm-like cooperation in budding yeast. Cell. 2008;135(4):726–37. 40. Tomás-Pejó E, Bonander N, Olsson L. Industrial yeasts strains for biore-finery solutions: constructing and selecting efficient barcoded xylose fermenting strains for ethanol. Biofuels Bioprod Bioref. 2014;8(5):626–34. 41. Wahlbom CF, van Zyl WH, Jönsson LJ, Hahn-Hägerdal B, Otero RRC. Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological compari-son with Pichia stipitis CBS 6054. FEMS Yeast Res. 2003;3(3):319–26. 42. van der Westhuizen TJ, Pretorius IS. The value of electrophoretic finger-printing and karyotyping in wine yeast breeding programmes. Antonie Van Leeuwenhoek. 1992;61(4):249–57. 43. Klimacek M, Kirl E, Krahulec S, Longus K, Novy V, Nidetzky B. Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae. Microb Cell Fact. 2014;13:37. 44. Novy V, Krahulec S, Wegleiter M, Muller G, Longus K, Klimacek M, Nidetzky B. Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three gen-erations of recombinant Saccharomyces cerevisiae. Biotechnol Biofuels. 2014;7:49. 45. Westman JO, Franzén CJ. A novel chimaeric flocculation protein improves flocculation characteristics in Saccharomyces cerevisiae. Submitted. 46. van Dijken JP, Bauer J, Brambilla L, Duboc P, Francois JM, Gancedo C, Giuseppin MLF, Heijnen JJ, Hoare M, Lange HC, Madden EA, Niederberger P, Nielsen J, Parrou JL, Petit T, Porro D. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol. 2000;26(9–10):706–14. 47. Gietz RD, Woods RA. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 2002;350:87–96. 48. Goldstein AL, McCusker JH. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 1999;15(14):1541–53. 49. Sluiter A, Hyman D, Payne C, Wolfe J. Determination of insoluble solids in pretreated biomass material. Laboratory analytical procedure (LAP) NREL/TP-510-42627. http://www.nrel.gov/docs/gen/fy13/42627.pdf. Issued 21 Mar 2008. National Renewable Energy Laboratory, 2008. 50. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of structural carbohydrates and lignin in biomass. Labora-tory analytical procedure (LAP) NREL/TP-510-42618. http://www.nrel.gov/docs/gen/fy13/42618.pdf. Issued 25 Apr 2008 (Revised 08/03/2012). 51. Verduyn C, Postma E, Scheffers WA, Van Dijken JP. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regula-tion of respiration and alcoholic fermentation. Yeast. 1992;8(7):501–17. 52. Adney B, Baker J. Measurement of cellulase activities: Laboratory Analyti-cal Procedure (LAP). NREL/TP-510-42628 1996, http://www.nrel.gov/docs/gen/fy08/42628.pdf. Issued 12 Aug 1996.Page 16 of 16Westman et al. Biotechnol Biofuels  (2017) 10:213 •  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step: 53. Basso LC, Basso TO, Rocha SN. Ethanol production in Brazil: the industrial process and its impact on yeast fermentation. In: Bernardes MAS, editor. Biofuel production - recent developments and prospects. Rijeka: InTech; 2011. p. 85–100. 54. Bothast RJ, Schlicher MA. Biotechnological processes for conversion of corn into ethanol. Appl Microbiol Biotechnol. 2005;67(1):19–25. 55. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G. Engineering yeast transcription machinery for improved ethanol tolerance and pro-duction. Science. 2006;314(5805):1565–8. 56. Albers E, Larsson C. A comparison of stress tolerance in YPD and industrial lignocellulose-based medium among industrial and laboratory yeast strains. J Ind Microbiol Biotechnol. 2009;36(8):1085–91. 57. Stratford M, Assinder S. Yeast flocculation: Flo1 and NewFlo phenotypes and receptor structure. Yeast. 1991;7(6):559–74. 58. Olofsson K, Bertilsson M, Lidén G. A short review on SSF - an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol Biofuels. 2008;1:7. 59. Jia K, Zhang Y, Li Y. Systematic engineering of microorganisms to improve alcohol tolerance. Eng Life Sci. 2010;10(5):422–9. 60. Lahtvee P-J, Kumar R, Hallström BM, Nielsen J. Adaptation to different types of stress converge on mitochondrial metabolism. Mol Biol Cell. 2016;27(15):2505–14. 61. Heipieper HJ, Weber FJ, Sikkema J, Keweloh H, de Bont JAM. Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol. 1994;12(10):409–15. 62. Mutturi S, Lidén G. Effect of temperature on simultaneous saccharifica-tion and fermentation of pretreated spruce and arundo. Ind Eng Chem Res. 2013;52(3):1244–51.


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items