Strategies for Reducing Supplemental Medium Cost in Bioethanol Production from Waste House Wood Hydrolysate by Ethanologenic Escherichia coli: Inoculum Size Increase and Coculture with Saccharomyces cerevisiae

doi:10.1263/jbb.105.90

Journal of Bioscience and Bioengineering

Volume 105, Issue 2, February 2008, Pages 90-96

https://doi.org/10.1263/jbb.105.90 Get rights and content

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor [CSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli KO11. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli KO11 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66–72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli KO11 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli KO11 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.

Section snippets

Microorganisms and media

Tryptone, yeast extract, and peptone were purchased from Becton, Dickinson and Company (Sparks, MD, USA). Chloramphenicol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Junsei Chemical (Tokyo). E. coli KO11, which was constructed by chromosomally integrating Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) and inactivating the pathway for succinate production (20), was supplied by Verenium Biofuel

Effect of CSL concentration on ethanol production by E. coli KO11

At first, to investigate the effect of CSL concentration on ethanol production by E. coli KO11 using the WHW hydrolysate medium, fermentation was conducted using the medium containing 1–4% CSL with 0.2 g-DCW/l E. coli KO11 as inoculum. As shown in Fig. 1, the xylose uptake ratio was 100%, and the overall ethanol yield reached 84% in the case of a CSL concentration of 4%. However, the xylose uptake ratio decreased to 40% when CSL concentration was 1%. The overall ethanol yield remained at 68% in

Discussion

Figure 5 shows the effect of generation number on volumetric ethanol production rate for various media containing E. coli KO11 and S. cerevisiae TJ1. For E. coli KO11 in 1%-CSL/hydrolysate medium, the volumetric ethanol production rate was approximately 0.4 g/(l·h) at the first generation; however, the rate decreased with an increase in the generation number, deteriorating to almost zero around the fifth to seventh generation. This indicates that treatment by overliming did not completely

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Screening and construction of a novel microbial consortium SSA-6 enriched from the gut symbionts of wood-feeding termite, Coptotermes formosanus and its biomass-based biorefineries
2019, Fuel
Biorefinery industry from lignocellulose faces one major obstacle which lies in the inefficient degradation of biomass. Lignocellulosic processes in wood-feeding termites are considered to be a very efficient biodegradation reaction due to a significant contribution from gut symbionts. In the present study, high degradation ratio of sawdust (84.3%) was achieved by the stable microbial consortium Cf-S, enriched from the termite gut symbionts of Coptotermes formosanus, within 120 h at 35 °C and pH 7. Molecular identification and phylogenetic analysis of Cf-S indicated that two yeasts and four bacterial candidates were identified for new species. Clustering and ordination analyses of Cf-S revealed that six strains Cf-S-2, Cf-S-6, Cf-S-11, Cf-S-20, Cf-S-22 and Cf-S-17 exhibited metabolic activities and physiological capabilities compared with other strains. These six strains were selected to construct a novel microbial consortium, designated SSA-6 that could make SSA-6 potentially consortium suitable for a wide variety of applications. The synergistic interaction of SSA-6 candidates led to significant higher cellulolytic, xylanolytic and ligninolytic activities than that produced by either strain alone (P = 0.003, 0.003 and 0.004, respectively). The consortium SSA-6 showed significant lower time for dye removal (Remazol Brilliant Blue R, Azure B, and Methylene Blue) together with higher dye concentration and decolorization ratio when compared with each individual strain (P < 0.001). The co-culture SSA-6 candidates produced 84.3, 203.6 and 168.4% more ethanol than that produced by Cf-S-2, Cf-S-17 and Cf-S-22 alone, respectively. This study shows the potential of constructing a novel microbial consortium valued for biomass-based biorefineries.
Development of cell recycle technology incorporating nutrient supplementation for lignocellulosic ethanol fermentation using industrial yeast Saccharomyces cerevisiae
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Citation Excerpt :
In contrast, the addition of CSL significantly improved the performance of yeast cells during CRBF, probably owing to the supplementation of proteins, amino acids, minerals, and vitamins present in CSL [30]. Compared with the results of the estimation of Okuda et al. [29], where 1% CSL was the criterion for decreasing the production cost (i.e., setting the cost of CSL to less than 10 yen per liter of ethanol), the results in Fig. 3 further decreased the required CSL concentration (<0.125% CSL). However, in the case of the low CSL concentration, there are considerable differences in ethanol concentration among the lignocellulosic materials used.
For the efficient production of lignocellulosic ethanol, a technology of recycling yeast cells at a high-solid load was developed. The yeast Saccharomyces cerevisiae TJ14 grew up to 5.0 × 10⁷ cells/mL at 39 °C during the simultaneous saccharification and fermentation of hydrothermally pretreated rice straw. By exploiting the viability of the cells, a portion of the fermentation residue was recycled, yielding 52.3 g/L ethanol (90.3% yield) at 1.12 g/(L·h) in six batches. This result is attributable to the maintenance of a viable cell count (cells/mL) on the order of 10⁷. In accordance with this principle, the effects of low-cost nutrients on yeast recycling were elucidated, where 0.125% corn steep liquor was deemed sufficient for fermenting rice straw and empty fruit bunch hydrolysates. The supplementation of sugarcane molasses into bagasse was found to be a practical approach, resulting in 63.5–67.7 g/L ethanol and 5.3 × 10⁶–4.4 × 10⁷ cells/mL in six batches. Here, we describe that the high cell-growth potential in industrially relevant media minimizes the retention of lignocellulosic residues in subsequent batches, thus eliminating the problematic operations in yeast recycling.
Solid state fermentative lignocellulolytic enzymes production, characterization and its application in the saccharification of rice waste biomass for ethanol production: An integrated biotechnological approach
2017, Journal of the Taiwan Institute of Chemical Engineers
Citation Excerpt :
Yeast co-culture utilized glucose and xylose efficiently; however the utilization of arabinose from RWB hydrolysate still remains poor. Okuda et al. [32], reported increase in ethanol yield from waste-house wood hydrolysate fermenting with the co-cultures of Escherichia coli K011 and S. cerevisiae TJ1. Similarly co-cultures of S. cerevisiae VS3 and P. stipitis NCIM 3498 showed maximum ethanol yield from Saccharum spontaneum hydrolysates reported earlier [11].
This work evaluates lignocellulolytic enzymes production by a selected strain Streptomyces sp. MDS cultivated in various agricultural wastes under solid-state fermentation (SSF). Maximum activities (U/gds) of endoglucanase (132.6 ± 1.15), exoglucanase (14.6 ± 0.62), cellobiase (125.6 ± 1.75), filter paperase (19.7 ± 0.42), amylase (278.5 ± 3.53), and xylanase (342.5 ± 3.36) were produced with rice waste biomass (RWB). Operational parameters and supplementation of nitrogen sources, metal additives, and surfactants were systematically optimized with a view to maximizing enzyme production. The harvested enzyme exhibited good stability at high pH (5 to 8) and temperature (50 to 80 °C) and showed robust nature in the presence of organic solvents, surfactants, and commercial detergents. The potential of crude enzyme mixture for hydrolysis of mild alkaline pretreated RWB was demonstrated, resulting in equivalent saccharification (64.85 ± 1.11%) relative to commercial enzymes (69.41 ± 1.15%). Yeast co-culture of Saccharomyces cerevisiae and Kluyveromyces marxianus resulted in higher ethanol production (21.12 ± 0.82 g/L) and sugar consumption (88%) from enzymatic RWB hydrolysates (50 g/L) than monoculture. Finally, the leftover spent slurry from SSF effectively decolorized individual dyes and the actual textile wastewater, which increases the practical applicability of the developed bioprocess.
In situ detoxification of dry dilute acid pretreated corn stover by co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae increases ethanol production
2016, Bioresource Technology
Citation Excerpt :
Co-culture fermentation, which means the use of two or multiple microorganisms simultaneous in one single fermenter, was often applied to achieve efficient conversion of both glucose and xylose. These systems include co-culture of immobilized Z. mobilis and free cells of Scheffersomyces stipitis (Fu et al., 2009; Karagöz and Özkan, 2014), co-culture of ethanologenic E. coli KO11 with S. cerevisiae (Okuda et al., 2008), co-culture of Z. mobilis and Candida tropicalis for ethanol production from hydrolyzed agricultural wastes (Patle and Lal, 2007), and co-culture of S. cerevisiae and Pachysolen tannophilus (Sathesh-Prabu and Murugesan, 2011). Simultaneous consumption of glucose, xylose, and arabinose was achieved by a consortium of E. coli strains (Xia et al., 2012).
Co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae was developed for bioethanol production from undetoxified pretreated biomass in simultaneously saccharification and co-fermentation (SSCF) process. Glucose accumulation during late fermentation phase in SSCF using xylose-utilizing strain can be eliminated by the introduction of inhibitor-tolerant strain. Effect of different ratios of two strains was investigated and xylose-utilizing strain to inhibitor-tolerant strain ratio of 10:1 (w/w) showed the best xylose consumption and the highest ethanol yield. Inoculating of xylose-utilizing strain at the later stage of SSCF (24–48 h) exhibited lower ethanol yield than inoculating at early stage (the beginning 0–12 h), probably due to the reduced enzymatic efficiency caused by the unconsumed xylose and oligomeric sugars. Co-culture SSCF increased ethanol concentration by 21.2% and 41.0% comparing to SSCF using individual inhibitor-tolerant and xylose-utilizing strain (increased from 48.5 and 41.7 g/L to 58.8 g/L), respectively, which suggest this co-culture system was very promising.
Effect of Saccharomyces cerevisiae and Zymomonas mobilis on the co-fermentation of sweet sorghum bagasse hydrolysates pretreated under varying conditions
2014, Biomass and Bioenergy
Hydrolysates from sweet sorghum bagasse pretreatment normally contains hexose and pentose sugars, and this complex mixture of sugars presents a challenge for a single microorganism to effectively ferment all sugars to ethanol. In this study, synergistic effects on the co-fermentation of the hydrolysates using Sacchromyces cerevisiae and Zymomonas mobilis ATCC31825 at different ratios were studied. An inoculum of mixed cultures (1:3 and 5:10 g/L, Z. mobilis to S. cerevisiae ratios) was investigated. Each mixed culture was added to the hydrolysates at pH 4.8 and incubated 32 °C for 24 h. The mixture of Z. mobilis to S. cerevisiae at 5:10 g/L showed the highest synergistic effect with ethanol yields of 0.5 g/g. Since the yield for co-culture was significantly higher than the sum of yields from each microorganism, the improvements can be directly related to co-fermentation of hydrolysate by S. cerevisiae and Z. mobilis.
Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass
2014, Biochemical Engineering Journal
Citation Excerpt :
First approach relied on the use of recombinant microorganism developed with the aim to ferment both xylose and glucose to ethanol. These include genetically modified strains of S. cerevisiae, Z. mobilis, Escherichia coli and Klebsiella oxytoca [7–12]. Though the high yield could be achieved from these genetically modified organisms (GMO's), their developmental cost, narrow and neutral pH range, long term stability and the utilization of residual cell mass as animal feed [4] like other GRAS organisms are the main disadvantages which restrict the use of GMO's at industrial level.
Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.

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Journal of Bioscience and Bioengineering

Section snippets

Microorganisms and media

Effect of CSL concentration on ethanol production by E. coli KO11

Discussion

References (22)

Fermentation of lignocellulosic hydrolysates I: inhibition and detoxification

Bioresour. Technol.

Fermentation of lignocellulosic hydrolysates II: inhibitors and mechanisms of inhibition. Bioresour

Technol.

DNA microarray analysis of the expression of the genes encoding the major enzymes in ethanol production during glucose and xylose co-fermentation by metabolically engineered Saccharomyces yeast

Enzyme Microb. Technol.

Kinetics of growth and ethanol production on different carbon substrates using genetically engineered xylose-fermenting yeast

Bioresour. Technol.

Ethanol production from a mixture of glucose and xylose by co-culture of Pichia stipitis and a respiratory-deficient mutant of Saccharomyces cerevisiae

J. Ferment. Bioeng.

Ethanol production from a mixture of glucose and xylose by a novel co-culture system with two fermentors and two microfiltration modules

J. Ferment. Bioeng.

Microaeration enhances productivity of bioethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11

J. Biosci. Bioeng.

Fuel ethanol production from lignocellulose: a challenge for metabolic enginneering and process integration

Appl. Microbiol. Biotechnol.

Detoxification of dilute acid hydrolysates of lignocellulose with lime

Biotechnol. Prog.

Relative rates of sugar utilization by an ethanologenic recombinant Escherichia coli using mixtures of glucose, mannose, and xylose

Appl. Biochem. Biotechnol.

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of l-lactic acid

J. Ind. Microbiol. Biotechnol.

Cited by (35)

Screening and construction of a novel microbial consortium SSA-6 enriched from the gut symbionts of wood-feeding termite, Coptotermes formosanus and its biomass-based biorefineries

Development of cell recycle technology incorporating nutrient supplementation for lignocellulosic ethanol fermentation using industrial yeast Saccharomyces cerevisiae

Solid state fermentative lignocellulolytic enzymes production, characterization and its application in the saccharification of rice waste biomass for ethanol production: An integrated biotechnological approach

In situ detoxification of dry dilute acid pretreated corn stover by co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae increases ethanol production

Effect of Saccharomyces cerevisiae and Zymomonas mobilis on the co-fermentation of sweet sorghum bagasse hydrolysates pretreated under varying conditions

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass