Batch Simultaneous Saccharification Research Articles

Ethanol production from the wood and fruit juice of paper mulberry via simultaneous saccharification and juice co-fermentation under normal and very high gravity conditions

A novel batch simultaneous saccharification and juice co-fermentation (SSJcF) process was investigated using mixtures of PHP (phosphoric acid plus hydrogen peroxide) pretreated wood and fruit juice of paper mulberry tree. All the different substrate mixture configurations evaluated under normal gravity SSJcF (total sugar titer of 180 g/L) significantly improved both ethanol concentration and yield compared to sole simultaneous saccharification and fermentation (SSF) of paper mulberry wood. Among the different mixing ratios, the feedstock mixture with equal sugar content from each substrate resulted in maximum ethanol concentration, yield and productivity of 77.1 g/L, 83.9 % and 1.1 g/L/h, respectively. The external additions of mineral nutrients also had no impact on fermentation performance of the mixture. Furthermore, SSJcF was evaluated under different levels of high gravity (HG) and very high gravity (VHG) conditions. The total sugar titer of 350 g/L at equal contributions from each substrate reached very high ethanol concentration of 147.0 g/L, with yield and productivity of 82.3 % and 1.5 g/L/h, respectively. By implication, the PM wood and fruit juice mixture could serve as suitable low-cost and non-food feedstock alternative to the typical use of sugary and/or starchy feedstocks for VHG fermentation.

Batch Simultaneous Saccharification and Fermentation of Primary Sludge at Very High Solid Concentrations for Bioethanol Production

A sustainable industrial future involves the exploitation of renewable resources to obtain a wide diversity of products and energy and the decrease of waste generation. Primary sludge (PS) from pulp and paper mills is a lignocellulosic residue mainly consisting of cellulose and hemicelluloses that can be converted to bioethanol. In the present work, bioethanol was produced from untreated PS by simultaneous saccharification and fermentation (SSF). Studies were carried out on initial solid concentration, yeast inoculum percentage, cellulolytic enzyme dosage, and co-application of two enzyme complexes (cellulolytic NS 22192 and xylanolytic Cellic® HTec2, Bagsværd, Denmark). Increasing solid content up to 22% improved ethanol concentration (59.1 g L−1), productivity (1.97 g L−1 h−1), and yield (86.3%); however, at the maximum solid concentration (28%), both yield and productivity decreased. At the highest solid concentration, a decrease of 33% in the cellulolytic enzyme dosage was observed (compared to reference enzyme loadings). The co-application of the two enzyme complexes had a positive effect on PS conversion efficiency. When a preliminary scale-up strategy was implemented from 50 mL to 2.5 L at 22% solids concentration, similar results were obtained despite the initial mixing difficulties of the heterogeneous system.

An aptly industrialized bioprocess for lactic acid production from corn stover using thermotolerant microbial consortia.

Chemical pretreatment of lignocellulosic biomass is a critical step in the conversion of lignocellulose to biofuels and biochemical. The main drawback of this pretreatment process is the formation of inhibitors which exhibit combined toxicity to microorganisms and result to low product concentrations and yields. In this study, the selection of microbial consortia by enrichment on hydrolysate of H2SO4-pretreated corn stover (pre-CS) without detoxification has been investigated as an efficient way to develop new strategies for lignocellulose utilization. The analysis of cattle stomach-dervied microbial consortia domesticated to degrade hydrolysate of pre-CS to produce lactic acid (LA) at different temperatures was investigated. Bacterial 16S rRNA gene amplicon sequencing analyses indicated that the three microbial consortia were taxonomically distinct and Enterococcus became dominant at high temperature. The highest glucose consumption rate was observed at 45°C, while the three microbial consortia showed similar consumption rates of xylose and arabinose. The selected microbial consortia DUT37, DUT45 and DUT47 showed preferable resistances to inhibitors in hydrolysate of pre-CS and abilities of xylose utilization. A batch simultaneous saccharification and fermentation (SSF) process was developed by microbial consortium DUT47 at 47°C to produce LA from pre-CS under non-detoxified and non-sterile conditions. The LA concentration and yield were 43.73g/L and 0.50g/g-corn stover (CS), respectively. Microbial consortium DUT47 has been shown to be suitable for LA production from H2SO4-pretreated corn stover without detoxification due to its thermophilic growth characteristics, robust tolerance of inhibitors, and the simultaneous utilization of glucose and xylose.

Intensive Multiple Sequential Batch Simultaneous Saccharification and Cultivation of Kluyveromyces marxianus SS106 Thermotolerant Yeast Strain for Single-Step Ethanol Fermentation from Raw Cassava Starch

We developed the intensive multiple sequential batch simultaneous saccharification and cultivation of the selected thermotolerant yeast strain for single-step ethanol production. The selection and high-cell-density inoculum production of thermotolerant yeast able to produce ethanol under the optimal conditions for single-step ethanol fermentation has become a necessity. In this study, the newly isolated Kluyveromyces marxianus SS106 could tolerate high temperatures (35–45 °C) and grow under a wide range of pH values (3.0–5.5), which are the optimum conditions of raw cassava starch hydrolyzing enzyme used in single-step ethanol fermentation. The high-cell-density concentration of K. marxianus SS106 was produced by a single batch and an intensive multiple sequential batch process in a 5-L stirred tank bioreactor using the simultaneous saccharification and cultivation (SSC) method. The single SSC process yielded the yeast cell biomass at a concentration of 39.30 g/L with a productivity of 3.28 g/L/h and a specific growth rate of 0.49 h−1. However, the yeast cell density concentration was higher in the intensive multiple sequential batch SSC than in the single batch process. This process yielded yeast cell biomass at concentrations of 36.09–45.82 g/L with productivities of 3.01–3.82 g/L/h and specific growth rates of 0.29–0.44 h−1 in the first six batch cycle. The results suggested that the intensive multiple sequential batch simultaneous saccharification and cultivation of K. marxianus SS106 would be a promising process for high-cell-density yeast production for use as the inoculum in single-step ethanol fermentation. Furthermore, we also experimented with single-step ethanol production from raw cassava starch by K. marxianus SS106 in a 5-L stirred tank fermenter. This produced ethanol at a concentration of 61.72 g/L with a productivity of 0.86 g/L/h.

Ethanol production from sugarcane bagasse by fed‐batch simultaneous saccharification and fermentation at high solids loading

AbstractTo make the bioethanol conversion process economically viable, improving solids loading (>15% [w/v]) in different unit operations is crucial. In this study, alkali pretreatment of sugarcane bagasse was carried out at high solids loading. Temperature and pH were optimised via batch simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae Y‐2034 or Kluyveromyces marxianus NCYC‐587. The effects of enzyme addition mode and solids loading on ethanol production were studied in fed‐batch SSF experiments. It was demonstrated that the optimised pH and temperature for both S. cerevisiae Y‐2034 and K. marxianus NCYC‐587 were 5.2 and 37°C. The mode of enzyme addition “one‐time dose” improved ethanol productivity better than the “batch addition” mode. With fresh substrates added, solids loading strongly increased from 19% to 33% (w/v), corresponded to a small decline in the ethanol yield. At the final solids loading of 33% (w/v), the highest ethanol concentration of 75.57 ± 2.14 g/L was obtained, with ethanol productivity of 0.63 ± 0.02 g/(L h) and a yield of 66.17 ± 1.87%.

Simultaneous saccharification and fermentation of sweet sorghum after acid pretreatment

ABSTRACTThe sweet sorghum bagasse pretreated with 5% (w/w) acetic acid at an accumulated solid concentration of 20% (w/v) during the 96-h fed-batch simultaneous saccharification and fermentation achieved a maximum ethanol concentration of 53.1 g/L and ethanol yield of 88.7%, compared to 25.7 g/L and 86.7% for the 96-h batch simultaneous saccharification and fermentation at a solid concentration of 10% (w/v), respectively. For comparison, the bagasse pretreated with 0.5% (w/w) sulfuric acid and water under the same fed-batch simultaneous saccharification and fermentation conditions produced maximum ethanol concentrations of 44.3 and 36.5 g/L, and ethanol yields of 77.6 and 69.7%, respectively.

PEI detoxification of pretreated spruce for high solids ethanol fermentation

Performing the bioethanol production process at high solids loading is a requirement for economic feasibility at industrial scale. So far this has successfully been achieved using wheat straw and other agricultural residues at 30% of water insoluble solids (WIS), but for softwood species (i.e. spruce) this has been difficult to reach. The main reason behind this difference is the higher recalcitrance of woody substrates which require harsher pretreatment conditions, thus generating higher amounts of inhibitory compounds, ultimately lowering fermentation performances. In this work we studied ethanol production from spruce performing the whole process, from pretreatment to hydrolysis and fermentation, at 30% dry matter (equivalent to ∼20% WIS). Hydrolysis and fermentation was performed in a horizontal free fall mixing reactor enabling efficient mixing at high solids loadings. In batch simultaneous saccharification and fermentation (SSF), up to 76% cellulose to ethanol conversion was achieved resulting in a concentration of 51g/kg of ethanol. Key to obtaining this high ethanol yield at these conditions was the use of a detoxification technology based on applying a soluble polyelectrolyte polymer (polyethylenimine, PEI) to absorb inhibitory compounds in the material. On average 50% removal and recovery of the main inhibitors (HMF, furfural, acetic acid and formic acid) was achieved dosing 1.5% w/w of soluble PEI. The use of PEI was compatible with operating the process at high solids loadings and enabled fermentation of hydrolysates, which was not possible without detoxification. In addition the possibility to recover the inhibitors and reuse PEI was studied.

Efficient utilization of cassava pulp for succinate production by metabolically engineered Escherichia coli KJ122.

A metabolically engineered Escherichia coli KJ122 was efficiently utilized for succinate production from cassava pulp during batch separate hydrolysis and fermentation (SHF) under simple anaerobic conditions. Succinate concentration of 41.46 ± 0.05 g/L with yield and productivity of 82.33 ± 0.14 g/100 g dry pulp and 0.84 ± 0.02 g/L/h was obtained. In batch simultaneous saccharification and fermentation (SSF), hydrolysis of 12 % (w/v) cassava pulp with an enzyme loading of 2 % AMG + 3 % Cel (v/w) at pH 6.5 was optimized at 39 °C. Succinate concentration of 80.86 ± 0.49 g/L with a yield of 70.34 ± 0.37 g/100 g dry pulp and a productivity of 0.84 ± 0.01 g/L/h was attained using E. coli KJ122. Fed-batch SSF significantly enhanced succinate concentration to 98.63 ± 0.12 g/L at yield and productivity of 71.64 ± 0.97 g/100 g dry pulp and 1.03 ± 0.01 g/L/h. This result indicated an efficient and economical succinate production from cassava pulp using SHF and SSF by the use of E. coli KJ122.

Optimal Temperature and pH Control for a Batch Simultaneous Saccharification and Co-Fermentation Process

Crisp and fuzzy optimization approaches were applied to design an optimal temperature and pH control policy for a batch process of simultaneous saccharification and co-fermentation (SSCF) for ethanol production from lignocellulose, using the enzyme and recombinant strain Zymomonas mobilis ZM (pZB5). To determine an optimal temperature and pH control policy, we applied the Arrhenius relationship to each rate constant to express the temperature and pH effects in the kinetic model for both saccharification and fermentation. The goal of the optimal design was to determine the optimal temperature, pH value, initial lignocellulosic concentration, and fermentation time for maximizing the ethanol productivity under the constraints of the follow-up separation specifications. The interactive crisp and fuzzy optimization methods were applied to solve the trade-off optimization problems for obtaining a compromised design. The fuzzy goal attainment approach obtained a compromised design more flexibly than did the crisp optimization. We also compared the performances for batch and fed-batch SSCF, and used various composition proportions for the batch SSCF to determine a series of optimal designs for the fuzzy goal attainment problem. Batch SSCF was slightly more effective than fed-batch fermentation, and spruce exhibited the maximum productivity because of its higher cellulose and lower hemicellulose contents compared with those of other sources.

Fed-batch SSCF using steam-exploded wheat straw at high dry matter consistencies and a xylose-fermenting Saccharomyces cerevisiae strain: effect of laccase supplementation

BackgroundLignocellulosic bioethanol is expected to play an important role in fossil fuel replacement in the short term. Process integration, improvements in water economy, and increased ethanol titers are key considerations for cost-effective large-scale production. The use of whole steam-pretreated slurries under high dry matter (DM) conditions and conversion of all fermentable sugars offer promising alternatives to achieve these goals.ResultsWheat straw slurry obtained from steam explosion showed high concentrations of degradation compounds, hindering the fermentation performance of the evolved xylose-recombinant Saccharomyces cerevisiae KE6-12 strain. Fermentability tests using the liquid fraction showed a higher number of colony-forming units (CFU) and higher xylose consumption rates when treating the medium with laccase. During batch simultaneous saccharification and co-fermentation (SSCF) processes, cell growth was totally inhibited at 12% DM (w/v) in untreated slurries. However, under these conditions laccase treatment prior to addition of yeast reduced the total phenolic content of the slurry and enabled the fermentation. During this process, an ethanol concentration of 19 g/L was obtained, corresponding to an ethanol yield of 39% of the theoretical yield. By changing the operation from batch mode to fed-batch mode, the concentration of inhibitors at the start of the process was reduced and 8 g/L of ethanol were obtained in untreated slurries with a final consistency of 16% DM (w/v). When fed-batch SSCF medium was supplemented with laccase 33 hours after yeast inoculation, no effect on ethanol yield or cell viability was found compared to untreated fermentations. However, if the laccase supplementation (21 hours after yeast inoculation) took place before the first addition of substrate (at 25 hours), improved cell viability and an increased ethanol titer of up to 32 g/L (51% of the theoretical) were found.ConclusionsLaccase treatment in SSCF processes reduces the inhibitory effect that degradation compounds have on the fermenting microorganism. Furthermore, in combination with fed-batch operational mode, laccase supplementation allows the fermentation of wheat straw slurry at high DM consistencies, improving final ethanol concentrations and yields.

The effect of prehydrolysis and improved mixing on high-solids batch simultaneous saccharification and fermentation of spruce to ethanol

In the production of ethanol from lignocellulosic material, it is necessary to reach a high ethanol concentration after fermentation. Simply increasing the substrate concentration leads to stirring problems and inhibition of the enzymes and yeast in the process.Batch simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce with 13.7% water-insoluble solids (WIS) (25% total solids (TS)) was run in a stirred-tank reactor as well as in two reactors designed to handle solid or semi-solid material. In all reactors, the overall ethanol yields were only between 5 and 6%. Fermentation of the liquid fraction of the steam-pretreated spruce slurry resulted in an overall ethanol yield of 85%.22h of prehydrolysis at 48°C prior to SSF at 32°C significantly increased the overall ethanol yield to 72% (final ethanol concentration of 47.8g/L), using the whole slurry of steam-pretreated spruce at a dry matter content of 13.7% WIS (25% TS).

CONCEPTUAL AND MATHEMATICAL MODELS OF BATCH SIMULTANEOUS SACCHARIFICATION AND FERMENTATION: DIMENSIONLESS GROUPS FOR PREDICTING PROCESS DYNAMICS

This paper describes a modeling effort demonstrating that dimensionless groupings of classical process parameters can be used to predicting process dynamics of batch simultaneous saccharification and fermentation (SSF) processes. Michaelis–Menten enzyme kinetics and Monod growth kinetics were employed, and inhibition of enzyme action and inhibition of microbial growth were neglected. The SSF process was characterized by the relative durations of three phases: A microbially-limited phase, a hydrolysis-limited phase, and a monosaccharide-depletion phase. The duration of these three phases were interrelated, and well predicted by the dimensionless magnitude of the monosaccharide peak (MSP). Thus, the MSP could be used as a single-value descriptor of an SSF process. The dimensionless ratio of the initial hydrolysis rate to the initial substrate consumption rate was shown to predict MSP, and an overall system time constant was shown to predict the total run time of a batch SSF process.

Simultaneous saccharification and co‐fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222—Part I: Kinetic modeling and parameters

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

Simultaneous Saccharification and Fermentation of Orange Processing Waste to Ethanol Using Kluyveromyces marxianus

Thermotolerant yeast Kluyveromyces marxianus performance was evaluated at elevated temperatures (37C to 45C) in batch simultaneous saccharification and fermentations (SSFs) using deterpenized high-solids (23.5% dry solids) orange processing waste (CPW) and a mixture of commercial pectinolytic, cellulolytic, and hemicellulolytic enzymes. Effects of inoculum size, temperature, pH, and nutritional supplements on ethanol production were studied. SSFs done at 40C 1C inoculated with K. marxianus cultures containing 0.126 0.027 mg cells g-1 CPW did not perform as well as SSFs with a 10 higher inoculation (1.26 0.27 mg cells g-1 CPW). Using the low inoculum, the highest ethanol production (2.5 wt%) was achieved with the addition of calcium carbonate sufficient to maintain pH above 4.8 and supplemented with ammonium sulfate or yeast extract . SSFs with the higher K. marxianus inoculum produced significantly higher ethanol concentrations of 3.4 to 3.5 wt% (84% to 86% theoretical yield) after 48 h for all treatments at 37C and for the lower carbonate concentrations (pH 4.0 to 4.2) at 40C. These results were equivalent to the ethanol produced (3.5 wt%, 86% theoretical yield) in SSFs using Saccharomyces cerevisiae at the same inoculum level after 48 h at 37C. Ethanol was also produced in SSFs at 42C and 45C with K. marxianus, but in significantly lower (18% to 38%) amounts. S. cerevisiae does not perform well above 37C, and K. marxianus may therefore be suitable for SSF of CPW in tropical and subtropical climates where citrus is processed and costs to cool and maintain temperatures below 35C are higher than in temperate regions.

Parameter Estimation for Simultaneous Saccharification and Fermentation of Food Waste Into Ethanol Using Matlab Simulink

The increase in waste disposal and energy costs has provided an incentive to convert carbohydrate-rich food waste streams into fuel. For example, dining halls and restaurants discard foods that require tipping fees for removal. An effective use of food waste may be the enzymatic hydrolysis of the waste to simple sugars and fermentation of the sugars to ethanol. As these wastes have complex compositions which may change day-to-day, experiments were carried out to test fermentability of two different types of food waste at 27 degrees C using Saccharomyces cerevisiae yeast (ATCC4124) and Genencor's STARGEN enzyme in batch simultaneous saccharification and fermentation (SSF) experiments. A mathematical model of SSF based on experimentally matched rate equations for enzyme hydrolysis and yeast fermentation was developed in Matlab Simulink. Using Simulink parameter estimation 1.1.3, parameters for hydrolysis and fermentation were estimated through modified Michaelis-Menten and Monod-type equations with the aim of predicting changes in the levels of ethanol and glycerol from different initial concentrations of glucose, fructose, maltose, and starch. The model predictions and experimental observations agree reasonably well for the two food waste streams and a third validation dataset. The approach of using Simulink as a dynamic visual model for SSF represents a simple method which can be applied to a variety of biological pathways and may be very useful for systems approaches in metabolic engineering in the future.

FED-BATCH SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF MICROWAVE/ACID/ALKALI/H2O2 PRETREATED RICE STRAW FOR PRODUCTION OF ETHANOL

ABSTRACT The batch simultaneous saccharification and fermentation (SSF) of microwave/acid/alkali/H2O2 pretreated rice straw to ethanol was optimized using cellulase from Trichoderma reesei and Saccharomyces cerevisiae YC-097 cells prior to the fed-batch SSF studies. The batch SSF optima were 10% w/v substrate, 40°C, 15 mg cellulase/g substrate, initial pH 5.3, and 72 hours. Under the optimum conditions the ethanol concentration and its yield were 29.1 g/L and 61.3% respectively. Based on the optimal batch SSF, the fed-batch SSF was investigated and its operation parameters were optimized. Under its optimal conditions the ethanol concentration reached 57.3 g/L, while its productivity and yield were only slightly less than those in the batch SSF. This suggests that fed-batch SSF is a potential operation mode for effective ethanol production from microwave/acid/alkali/H2O2 pretreated rice straw.

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).