Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings
© Koppram and Olsson; licensee BioMed Central Ltd. 2014
Received: 24 December 2013
Accepted: 13 March 2014
Published: 8 April 2014
Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings.
In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading.
A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a conventional bioreactor. The proposed process strategy significantly increased the yeast cell viability and overall ethanol yield. It was also possible to obtain 4% (w/v) ethanol concentration, which is a minimum requirement for an economical distillation process.
KeywordsHigh solids High gravity Saccharomyces cerevisiae SSF
Bioethanol produced from lignocellulosic raw materials is considered as a potential transportation fuel providing long-term energy security as well as environmental and economical benefits . Biological conversion of carbohydrates in lignocelluloses to ethanol can be realized by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) of the pretreated raw material. Reduced number of process reactors is one of the features of SSF, which integrates enzymatic hydrolysis and fermentation in one reactor. In SSF, the released sugars from enzymatic hydrolysis are simultaneously consumed by the fermenting microorganism, for example, Saccharomyces cerevisiae during fermentation avoiding product inhibition of enzymes and also decreasing the probability of contamination . Distillation of ethanol from the fermentation broth is one of the energy intensive steps  and it is crucial to achieve the highest possible ethanol concentration in the fermentation broth, because the cost of distillation decreases with increase in ethanol concentration . Ethanol concentration of 4% (w/v) is the minimal requirement for an economical distillation process. By increasing the water insoluble solids (WIS) concentration in an SSF process, it is possible to achieve high sugar concentration and consequently high final ethanol concentration. Currently, when operating at a high WIS content in conventional stirred tank reactors technological challenges remain, which include high viscosity preventing efficient mixing, high power consumption  and high concentrations of lignocelluloses-derived inhibitors [6, 7] that inhibit cellulolytic enzymes and metabolism of S. cerevisiae. A detailed review of the challenges encountered at high solids loading, its pervasive effect on the pretreatment, enzymatic hydrolysis and fermentation has been presented by Koppram et al..
Inhomogeneity caused by inadequate mixing has been previously addressed in several ways. Different reactor designs such as a liquefaction reactor  and a simple rotary fermenter  have been designed and fabricated and the SSF functionality of these has been demonstrated using pretreated wheat straw with a dry matter content of 32% (w/w) and higher. However, design of specialized reactors is often expensive and may restrict the functionality to a particular feedstock. Alternatively, using conventional stirred-tank reactors several groups have performed SSF in fed-batch mode with substrate and/or enzyme feed [11, 12] to overcome challenges when working with high WIS content. A comprehensive review of SSF has been presented by Olofsson et al. . Fed-batch SSF has been shown on many occasions to be beneficial for various aspects including: (a) ease of mixing after partial saccharification, resulting in the capacity for more substrate to be added in a stepwise procedure ; (b) lower energy consumption due to lower viscosity  at any given time point compared to batch SSF; (c) low concentration of inhibitory compounds facilitating the yeast, S. cerevisiae to convert them to less inhibitory compounds , and (d) maintaining low glucose concentration in the medium, facilitating effective co-consumption of glucose and xylose by recombinant S. cerevisiae[16, 17]. The potential of fed-batch SSF with substrate and enzyme feed using recombinant xylose utilizing S. cerevisiae, at a demo scale of a 10-m3 conventional bioreactor, has also been demonstrated using 10% (w/w) WIS content of pretreated corn cobs, producing 4% (w/v) ethanol . Although substrate and enzyme feeding strategies in SSF have been widely explored, the significance of yeast feed in an SSF process remains to be investigated. One of the elemental parts of SSF is the yeast, S. cerevisiae, which at high WIS content is subjected to high concentration of inhibitors that affect cell viability [19, 20], growth, ethanol yield and productivity [21–23]. Although several detoxification methods can be employed to partly remove the inhibitors , the cost of such methods limits their use . Attempts at process modifications to curb the effects of inhibitors have been fruitful. It has been shown that prior to SSF, cultivating the cells in fed-batch mode using the liquid fraction derived from pretreatment improved tolerance towards inhibitors and ethanol productivity in an SSF process using pretreated spruce of relatively low WIS content of 8% (w/w) . However, when working at WIS content as high as 20% (w/w), the severity of inhibition increases and therefore, maintaining the viability of yeast throughout the SSF process becomes crucial. In the present work, we developed a continuous mode of cultivation for adaptation, wherein the outflow of cell suspension from the adaptation reactor was fed to the SSF reactor with the objective of maintaining the robustness of yeast cells during the SSF process. With this mode of yeast feed together with the substrate and enzyme-cocktail feed we evaluated the performance of SSF at 20% (w/w) WIS content using pretreated spruce as biomass. In a parallel study, a mathematical model for the SSF process has been developed and the effect of substrate, enzyme and cell feeding was analyzed (Wang R, Koppram R, Olsson L and Franzén C-J. Modeling and experimental studies of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol. Manuscript).
Results and discussion
The aim of the current study was to design an SSF process of high WIS content in a conventional stirred-tank bioreactor. We approached this challenge by using a combination of yeast, enzyme and substrate feed as a means to improve the fermentability at high WIS. In addition, we designed a continuous process for cultivation and adaptation of the yeast stream, to allow optimum performance of the S. cerevisiae Thermosacc.
Evaluation of fermentation performance of Thermosacc
Composition of spruce slurry and non-washed solids used in simultaneous saccharification and fermentation (SSF)
Concentration when slurry was used, g kg-1*
Concentration when non-washed solids were used, g kg-1*
SSF with the solid fraction
High WIS content in batch SSF causes inhomogeneity
Yeast feed improves the overall ethanol yield in SSF at high WIS content
Summary of simultaneous saccharification and fermentation (SSF) experiments
Water insoluble solids (WIS)
Yeast loading amount, g
Enzyme loading, FPU
10 to 20b
50 to 300
1125 to 2250d
10 to 20b
50 to 300
3.5 to 7.5c
10 to 20b
50 to 300
3.5 to 7.5c
1125 to 2250d
Although the substrate feed improved the mixing and cell feed improved the cell viability, the highest observed ethanol yield was only 53% of the theoretical maximum despite the fact that a large fraction of cells remained viable even after 96 h of fed-batch SSF (Figure 4). The concentration levels of hexose sugars including glucose, mannose and galactose remained below 0.3 g L-1 after 96 h, indicating that there were no fermentable monomeric sugars. This likely indicates that enzymatic hydrolysis could be a possible limiting factor affecting the overall ethanol yield. It has previously been shown that at increasing solids concentration, the proportion of adsorbed cellulase decreased because of adsorption inhibition, the mechanism of which remains elusive . Also, there is previous evidence that xylose and xylooligomers with a concentration as low as 1.67 g L-1 can decrease enzymatic hydrolysis rates and yields . In our study xylose was not fermentable by Thermosacc and the existence of xylose at a significant concentration of 15 g L-1 in the SSF can hinder enzymatic hydrolysis. Besides, softwoods, such as spruce, contain relatively high lignin content , which can cause increased nonspecific adsorption of cellulases to lignin  especially at high WIS content. Evidence also suggests that the simple and oligomeric phenolics generated during pretreatment, cause inhibition and precipitation of enzymes, even at low concentrations [6, 33]. Furthermore, the operation of SSF at suboptimal temperature of enzymes combined with other aforementioned factors are some of the areas that need improvement to further increase the ethanol yields to make the process economically feasible.
We here demonstrated that the cultivation of yeast in a continuous culture wherein the outflow of cell suspension was fed to the SSF reactor, can significantly enhance cell viability and contribute to overall increase in ethanol yield. We also show that it is possible to work at a high WIS content of 20% (w/w) in conventional bioreactors using a well-designed fed-batch SSF process. Furthermore, we demonstrated the production of high ethanol-concentration (40 g L-1), which is the minimal requirement for an economical distillation process. In addition, the potential of fed-batch SSF with substrate, enzyme and yeast feed can be improved by addressing the challenges pertaining to enzymatic hydrolysis.
The inoculum for anaerobic fermentations and SSF experiments was prepared by cultivation in minimal medium containing 20 g L-1 glucose and enriched with salts, and 2-fold addition of vitamins and trace elements according to Verduyn et al.. The pH of the medium was set to 6.0 with 1 M NaOH for all shake-flask cultivations. YPD plates containing 10 g L-1 yeast extract, 20 g L-1 peptone, 20 g L-1 glucose and 20 g L-1 agar were used for colony forming unit (CFU) determination during the SSF. A YNB plate containing 6.9 g L-1 yeast nitrogen base (without amino acids), 20 g L-1 glucose and 20 g L-1 agar was used to isolate individual colonies of yeast.
S. cerevisiae Thermosacc Dry was purchased from Lallemand, USA. The dry yeast was suspended in 5 ml of minimal medium and a loop full of cell suspension was streaked on a YNB plate, which was later incubated at 30°C for 2 days. A loop full of colonies of the same size were picked and re-suspended in 50 ml of minimal medium in a 150-ml shake flask, which was later incubated at 30°C on an orbital shaker set at 180 rpm until the late exponential phase (approximately 20 h). Aliquots of cell suspension (1 ml) were mixed with 0.5 ml of 60% sterile glycerol and stored at -80°C in sterile vials. Volumes of 100 μl from the vials were used to inoculate precultures.
Spruce slurry with a WIS content of 20.3%, w/w (weight of insoluble solids to weight of slurry) was received from SEKAB-E-Technology AB (Örnsköldsvik, Sweden) and the composition of slurry is given in Table 1. The slurry was centrifuged at 10,000 g for 10 minutes to separate the solid and the liquid fractions. Neither the solid nor the liquid fraction was subjected to chemical or physical detoxification. The solid fraction was used as a substrate feed for SSF. The liquid fraction along with the minimal medium was used for cultivation during the adaptation step. The liquid fraction was also used for anaerobic fermentation to assess its fermentability by Thermosacc.
Cultivation of Thermosacc
The preculture for cell cultivation was developed in 50 ml of minimal medium in a 150-ml shake flask incubated at 30°C on an orbital shaker set at 180 rpm for 20 h. The cells were cultivated in a 3.6-L Infors HT-Labfors bioreactor in two stages, an initial batch phase in minimal medium, followed by a fed-batch phase of adaptation in a medium of liquid fraction with minimal medium. The batch phase was initiated by adding 50 ml of preculture to a working volume of 500 ml, and the cultivation was carried out until the growth on glucose followed by ethanol was completed, which was indicated by CO2 evolution in the off-gas and by the dissolved oxygen concentration in the culture. Upon exhaustion of glucose and ethanol in the batch phase, a feed solution of the liquid fraction (from pretreated slurry) and minimal medium was fed linearly for 16 h to a final volume of 1.3 L. The concentration of liquid fraction in the feed solution was 50% (v/v). The minimal medium was supplemented to the feed solution to a final hexose (glucose, mannose and galactose) concentration of 50 g L-1 and the salts, vitamins and trace elements were correspondingly scaled up. The stirrer speed was set to 700 rpm during the batch phase and increased linearly to 1,000 rpm during the fed-batch phase. The aeration rate was maintained at 1 volume per volume per minute (vvm) and the pH was maintained at 5.0 by automatic addition of 2 M NaOH. Cell suspension was harvested after the fed-batch phase and used as yeast loading for anaerobic fermentation and also as initial yeast loading for the subsequent fed-batch SSF process. After the fed-batch phase the cultivation process was changed to a continuous mode with the adaptation feed solution with a flow rate of 50 ml h-1 and the working volume was maintained at 1 L. A part of the outflow was manually regulated as a cell suspension feed to the SSF reactor.
Anaerobic fermentation in shake flasks
The fermentation was carried out to a working volume of 50 ml in 100-ml shake flasks fitted with a glycerol loop providing an anaerobic condition. Different concentrations of the liquid fraction (90%, 60% and 40% (v/v)) were screened. The pH of the liquid fraction was adjusted to 6.0 and supplemented with 0.5 g l-1 (NH4)2HPO4, 125 ppm of vitahop (Betatech Gmbh, Schwabach, Germany) (to suppress bacterial growth). The fermentation was initiated by adding harvested cell suspension to reach a yeast concentration of 3 g dry weight L-1. The flasks were incubated at 30°C on an orbital shaker set at 180 rpm for 96 h and samples were withdrawn for optical density (OD)650 measurement and extracellular metabolite analysis.
All the SSF experiments were carried out to a total WIS content of 20% (w/w), total enzyme loading of 7.5 FPU g-1 WIS, total yeast loading of 5 g dry weight L-1 and to a final working weight of 1.5 kg in 3.6-L Infors HT-Labfors reactors. An enzyme preparation, Cellic Ctec-2 from Novozymes A/S, Denmark with a filter paper activity of 149 FPU g-1 enzyme, β-glucosidase activity of 590 IU g-1 enzyme was used. The solid fraction of the pretreated spruce was used as the substrate, and the pH of this was adjusted to 5.0 using 10 M NaOH and supplemented with 0.5 g L-1 (NH4)2HPO4 and 125 ppm of vitahop. Batch SSF was initiated by adding fed-batch adapted cell suspension and enzyme preparation. Fed-batch SSF was initially started as a batch SSF of 500 g as a working weight with 10% (w/w) WIS content, 50% of total yeast cell suspension (fed-batch adapted) and 50% of total enzyme preparation. After an initial period of 8 h of batch SSF the solid fraction, enzyme preparation and yeast cell suspension (continuous mode adapted) were manually fed every 4 or 8 h. The manual feeding was carried out for a period of 65 h. Fed-batch SSF with substrate and enzyme-cocktail feed was carried out in a similar way but with all the required yeast cell suspension added at the beginning. Fed-batch SSF with substrate and cell-suspension feed was carried out in a similar manner but with all the required enzyme preparation added at the beginning. The temperature was maintained at 35°C and pH at 5.0 by automatic addition of 5 M NaOH. The stirrer speed was set at 700 rpm for batch SSF and 400 rpm for fed-batch SSF. All the experiments were carried out in duplicates.
Samples collected during the SSF were serially diluted using sterile normal saline solution; 100 μl of two of the dilutions were plated on YPD plates and incubated at 30°C for 2 days and the colonies were counted and represented as CFU ml-1.
To make the sample pipettable equivalent to water, 1 g of the withdrawn sample from SSF was diluted five times with milliQ water. The diluted samples were quantified for metabolites by HPLC. The concentrations (including the dilution factor) of metabolites obtained by HPLC were represented in g (metabolite) kg (slurry)-1. This concentration was used to determine the yields presented in Table 2. However, for the data representation in the graphs and elsewhere in the text, the concentration (g kg-1) was converted to concentration (g L-1) by multiplying with a conversion factor of 1.06 (kg L-1). The conversion factor was determined by pipetting 1 ml of slurry using a cut tip and weighing.
Analysis of metabolites
Samples for extracellular metabolites were analyzed by HPLC using Aminex HPX-87H column with 30 × 4.6 mm Cation-H Biorad micro-guard column (Bio-Rad Laboratories AB, Solna, Sweden) maintained at 45°C; 5 mM H2SO4 was used as an eluent at a flow rate of 0.6 ml min-1. Glycerol, ethanol and acetic acid were detected using an RI detector maintained at 35°C; HMF and furfural were detected using a UV detector at 210 nm. The monomeric sugars in the samples were analyzed by high performance anion exchange chromatography using 4 × 250 mm Dionex CarboPac PA1 column with 4 × 50 mm guard column (Thermo Scientific, Sweden) maintained at 30°C. Eluent A: 300 mM NaOH, eluent B: 100 mM NaOH + 85 mM sodium acetate were used for elution at a flow rate of 1 ml min-1. Monosaccharides including galactose, glucose and mannose were detected using pulsed amperometric detector.
Ethanol yield calculation
filter paper units
high performance anion exchange chromatography
high performance liquid chromatography
simultaneous saccharification and fermentation
water insoluble solids
yeast extract peptone dextrose.
This work was a part of the project ‘High Gravity Biofuels’ funded by Top level Nordic Research Institute. We thank SEKAB-E-Technology AB for providing us with pretreated spruce. The authors thank Associate Professor Carl Johan Franzén, Dr Charilaos Xiros and Ruifei Wang for the valuable discussion.
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