Consolidated Bioprocessing for Bioethanol Production by Metabolically Engineered Bacillus Subtilis Strains

2021 ◽  
Author(s):  
Fatemeh Maleki ◽  
Mohammad Changizian ◽  
Narges Zolfaghari ◽  
Sarah Rajaei ◽  
Kambiz Akbari Noghabi ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Plant Biomass ◽  
Fusion Gene ◽  
Consolidated Bioprocessing ◽  
Gene Dosage ◽  
Pyruvate Decarboxylase ◽  
Single Step ◽  
Bioethanol Production ◽  
Genes Encoding

Abstract Background: Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. Results: In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. Conclusion: In this study for the first time, Bacillus subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

scholarly journals Single-step ethanol production from raw cassava starch using a combination of raw starch hydrolysis and fermentation, scale-up from 5-L laboratory and 200-L pilot plant to 3000-L industrial fermenters

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Morakot Krajang ◽  
Kwanruthai Malairuang ◽  
Jatuporn Sukna ◽  
Krongchan Rattanapradit ◽  
Saethawat Chamsart
Keyword(s):  
Ethanol Production ◽  
Ethanol Concentration ◽  
Consolidated Bioprocessing ◽  
Scale Up ◽  
Cassava Starch ◽  
Single Step ◽  
Starch Hydrolysis ◽  
Yield Coefficient ◽  
Raw Starch ◽  
Raw Starch Hydrolysis

Abstract Background A single-step ethanol production is the combination of raw cassava starch hydrolysis and fermentation. For the development of raw starch consolidated bioprocessing technologies, this research was to investigate the optimum conditions and technical procedures for the production of ethanol from raw cassava starch in a single step. It successfully resulted in high yields and productivities of all the experiments from the laboratory, the pilot, through the industrial scales. Yields of ethanol concentration are comparable with those in the commercial industries that use molasses and hydrolyzed starch as the raw materials. Results Before single-step ethanol production, studies of raw cassava starch hydrolysis by a granular starch hydrolyzing enzyme, StargenTM002, were carefully conducted. It successfully converted 80.19% (w/v) of raw cassava starch to glucose at a concentration of 176.41 g/L with a productivity at 2.45 g/L/h when it was pretreated at 60 °C for 1 h with 0.10% (v/w dry starch basis) of Distillase ASP before hydrolysis. The single-step ethanol production at 34 °C in a 5-L fermenter showed that Saccharomyces cerevisiae (Fali, active dry yeast) produced the maximum ethanol concentration, pmax at 81.86 g/L (10.37% v/v) with a yield coefficient, Yp/s of 0.43 g/g, a productivity or production rate, rp at 1.14 g/L/h and an efficiency, Ef of 75.29%. Scale-up experiments of the single-step ethanol production using this method, from the 5-L fermenter to the 200-L fermenter and further to the 3000-L industrial fermenter were successfully achieved with essentially good results. The values of pmax,Yp/s, rp, and Ef of the 200-L scale were at 80.85 g/L (10.25% v/v), 0.42 g/g, 1.12 g/L/h and 74.40%, respectively, and those of the 3000-L scale were at 70.74 g/L (8.97% v/v), 0.38 g/g, 0.98 g/L/h and 67.56%, respectively. Because of using raw starch, major by-products, i.e., glycerol, lactic acid, and acetic acid of all three scales were very low, in ranges of 0.940–1.140, 0.046–0.052, 0.000–0.059 (% w/v), respectively, where are less than those values in the industries. Conclusion The single-step ethanol production using the combination of raw cassava starch hydrolysis and fermentation of three fermentation scales in this study is practicable and feasible for the scale-up of industrial production of ethanol from raw starch.

scholarly journals Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Tristan Cerisy ◽  
Tiffany Souterre ◽  
Ismael Torres-Romero ◽  
Magali Boutard ◽  
Ivan Dubois ◽  
...  
Keyword(s):  
Ferulic Acid ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Gene Dosage ◽  
Content Type ◽  
Chemical Inhibitors ◽  
Growth Selection ◽  
Clostridium Phytofermentans

ABSTRACT Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors. IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.

Effects of manipulation of pyruvate decarboxylase and alcohol dehydrogenase levels on the submergence tolerance of rice

2001 ◽  
Vol 28 (12) ◽  
pp. 1231 ◽  
Author(s):  
Musrur Rahman ◽  
Anil Grover ◽  
W. James Peacock ◽  
Elizabeth S. Dennis ◽  
Marc H. Ellis
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Rice Variety ◽  
Pyruvate Decarboxylase ◽  
Inducible Promoter ◽  
Moderate Increase ◽  
Sense Orientation ◽  
Transgenic Lines ◽  
Hybrid Lines ◽  
Adh Activity

A transgenic approach was taken to manipulate the levels of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in rice, in order to investigate whether alteration of ethanol fermentation can affect anaerobic tolerance. A line transformed with an antisense Adh1 construct had only 4–8% of the ADH activity of untransformed plants. This line showed reduced ethanol production and coleoptile growth under anoxia. Mature plants had reduced survival when submerged in water and exposed to anoxia, suggesting that ADH plays an essential role in seed germination and plant survival in the absence of O2. A transgenic line transformed with a cotton Adh2 cDNA in the sense orientation relative to a constitutive promoter, showed 3–4-fold more ADH activity than either untransformed controls, or a flooding-tolerant rice variety (FR13A), both in air and under hypoxia. However, ethanol production by this line was only slightly higher than that of untransformed controls, and there was no increase in survival following anoxia treatments. Three independent transgenic lines containing the ricePdc1 cDNA driven by an anaerobically-inducible promoter (6XARE) showed an increase in PDC1 polypeptide in shoots, but not in roots or endosperm. A moderate increase in PDC activity and ethanol production was observed in shoots of these lines under anaerobic conditions, as well as decreased survival of shoots when submerged and exposed to anoxia. F1 plants containing both the PDC and ADH constructs showed levels of anoxia-tolerance similar to those of untransformed plants. These results suggest that over-production of PDC may be toxic to rice plants because of increased acetaldehyde. Consistent with this view, acetaldehyde levels were appreciably higher in plants over-producing PDC, compared with untransformed plants, or hybrid lines containing both the PDC and ADH constructs.

Fusion of Pyruvate Decarboxylase and Alcohol Dehydrogenase Increases Ethanol Production inEscherichia coli

2014 ◽  
Vol 3 (12) ◽  
pp. 976-978 ◽  
Author(s):  
Aleksandra J. Lewicka ◽  
Jan J. Lyczakowski ◽  
Gavin Blackhurst ◽  
Christiana Pashkuleva ◽  
Kyle Rothschild-Mancinelli ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Pyruvate Decarboxylase ◽  
Inescherichia Coli

scholarly journals Zymobacter palmae pyruvate decarboxylase is less efficient than that of Zymomonas mobilis for ethanol production in metabolically engineered Synechocystis sp PCC6803

2019 ◽  
Author(s):  
Lorraine Quinn ◽  
Patricia Armshaw ◽  
Tewfik Soulimane ◽  
Con Sheehan ◽  
Michael P Ryan ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Inducible Promoter ◽  
Control Strain ◽  
Zymobacter Palmae ◽  
Synechocystis Sp ◽  
Pcc 6803

AbstractPyruvate decarboxylase (PDC) from Zymobacter palmae (ZpPDC) has been reported to have a lower Km the Zymomonas mobilis PDC (ZmPDC). ZpPDC was combined with native slr1192 alcohol dehydrogenase (adh) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with Zmpdc. Native (Zppdc) and codon optimised (ZpOpdc) versions of the ZpPDC were cloned into a construct where the pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis PCC 6803. These constructs were transformed into wildtype Synechocystis PCC 6803. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL004) expressing the pdc from Zymomonas mobilis. The utilisation of a PDC with a lower Km from Zymobacter palmae did not result in enhanced ethanol production in Synechocystis PCC 6803.

scholarly journals METABOLIC CHANGES ASSOCIATED WITH OXYGEN STRESS IN PEAR FRUITS

HortScience ◽  
1990 ◽  
Vol 25 (11) ◽  
pp. 1355F-1356
Author(s):  
George D. Nanos ◽  
Roger J. Romani ◽  
Adel A. Kader
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethylene Production ◽  
Pyruvate Decarboxylase ◽  
Insect Control ◽  
Pear Fruit ◽  
Oxygen Stress ◽  
Fruit Tissue ◽  
Subsequent Recovery ◽  
Alcohol Dehydrogenase Activity

The response of pear fruits and suspension-cultured pear fruit cells to 0% or 0.25% O2 is being examined to evaluate the feasibility of using such atmospheres for postharvest insect control. These treatments inhibited ethylene production, had no effect on acetaldehyde content, and increased ethanol production in pears kept at 20C for 10 days. The blossom end area of pear fruits was more prone to anaerobiosis, as indicated by increased alcohol dehydrogenase activity and ethanol content. Pear fruit plugs showed increased respiration and ethylene production rates when skin was present compared to plugs without skin. Methods for measuring activity of alcohol dehydrogenase, pyruvate decarboxylase, and pyruvate kinase have been modified and optimized and will be used to determine changes in pear fruit tissue during low O2 treatment and subsequent recovery in air.

scholarly journals Consolidated Bioprocess for Bioethanol Production from Raw Flour of Brosimum alicastrum Seeds Using the Native Strain of Trametes hirsuta Bm-2

2019 ◽  
Vol 7 (11) ◽  
pp. 483 ◽  
Author(s):  
Olguin-Maciel ◽  
Larqué-Saavedra ◽  
Lappe-Oliveras ◽  
Barahona-Pérez ◽  
Alzate-Gaviria ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Consolidated Bioprocessing ◽  
Cost Effective ◽  
Raw Material ◽  
Ethanol Stress ◽  
Bioethanol Production ◽  
Trametes Hirsuta ◽  
Native Strain ◽  
Consolidated Bioprocess ◽  
Brosimum Alicastrum

Consolidated bioprocessing (CBP), which integrates biological pretreatment, enzyme production, saccharification, and fermentation, is a promising operational strategy for cost-effective ethanol production from biomass. In this study, the use of a native strain of Trametes hirsuta (Bm-2) was evaluated for bioethanol production from Brosimum alicastrum in a CBP. The raw seed flour obtained from the ramon tree contained 61% of starch, indicating its potential as a raw material for bioethanol production. Quantitative assays revealed that the Bm-2 strain produced the amylase enzyme with activity of 193.85 U/mL. The Bm-2 strain showed high tolerance to ethanol stress and was capable of directly producing ethanol from raw flour at a concentration of 13 g/L, with a production yield of 123.4 mL/kg flour. This study demonstrates the potential of T. hirsuta Bm-2 for starch-based ethanol production in a consolidated bioprocess to be implemented in the biofuel industry. The residual biomass after fermentation showed an average protein content of 22.5%, suggesting that it could also be considered as a valuable biorefinery co-product for animal feeding.

scholarly journals Zymobacter palmae Pyruvate Decarboxylase is Less Effective Than That of Zymomonas mobilis for Ethanol Production in Metabolically Engineered Synechocystis sp. PCC6803

2019 ◽  
Vol 7 (11) ◽  
pp. 494 ◽  
Author(s):  
Lorraine Quinn ◽  
Patricia Armshaw ◽  
Tewfik Soulimane ◽  
Con Sheehan ◽  
Michael P. Ryan ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Biomass Productivity ◽  
Gene Cassette ◽  
Major Effect ◽  
Zymobacter Palmae ◽  
Synechocystis Sp ◽  
Pcc 6803

To produce bioethanol from model cyanobacteria such as Synechocystis, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.

scholarly journals Single-Step Ethanol Production from Raw Cassava Starch Using a Combination of Raw Starch Hydrolysis and Fermentation, Scale-Up from 5-L Laboratory and 200-L Pilot Plant to 3,000-L Industrial Fermenters

2020 ◽  
Author(s):  
Morakot Krajang ◽  
Kwanruthai Malairuang ◽  
Jatuporn Sukna ◽  
Krongchan Rattanapradit ◽  
Saethawat Chamsart
Keyword(s):  
Ethanol Production ◽  
Ethanol Concentration ◽  
Consolidated Bioprocessing ◽  
Scale Up ◽  
Cassava Starch ◽  
Single Step ◽  
Starch Hydrolysis ◽  
Yield Coefficient ◽  
Raw Starch ◽  
Raw Starch Hydrolysis

Abstract Background: A single-step ethanol production is the combination of raw cassava starch hydrolysis and fermentation. For the development of raw starch consolidated bioprocessing technologies, this research was to investigate the optimum conditions and technical procedures for the production of ethanol from raw cassava starch in a single step. It successfully resulted in high yields and productivities of all the experiments from the laboratory, the pilot, through the industrial scales. Yields of ethanol concentration are comparable with those in the commercial industries that use molasses and hydrolyzed starch as the raw materials.Results: Before single-step ethanol production, studies of raw cassava starch hydrolysis by a granular starch hydrolyzing enzyme, StargenTM002, were carefully conducted. It successfully converted 80.19% (w/v) of raw cassava starch to glucose at a concentration of 176.41 g/L with a productivity at 2.45 g/L/h when it was pretreated at 60 °C for 1 h with 0.10% (v/w dry starch basis) of Distillase ASP before hydrolysis. The single-step ethanol production at 34 °C in a 5-L fermenter showed that Saccharomyces cerevisiae (Fali, active dry yeast) produced the maximum ethanol concentration, pmax at 81.86 g/L (10.37% v/v) with a yield coefficient, Yp/s of 0.43 g/g, a productivity or production rate, rp at 1.14 g/L/h and an efficiency, Ef of 75.29%. Scale-up experiments of the single-step ethanol production using this method, from the 5-L fermenter to the 200-L fermenter and further to the 3,000-L industrial fermenter were successfully achieved with essentially good results. The values of pmax, Yp/s, rp, and Ef of the 200-L scale were at 80.85 g/L (10.25% v/v), 0.42 g/g, 1.12 g/L/h and 74.40%, respectively and those of the 3,000-L scale were at 70.74 g/L (8.97% v/v), 0.38 g/g, 0.98 g/L/h and 67.56%, respectively. Because of using raw starch, major by-products, i.e., glycerol, lactic acid, and acetic acid of all three scales were very low, in ranges of 0.940−1.140, 0.046−0.052, 0.000−0.059 (% w/v), respectively, where are less than those values in the industries.Conclusion: The single-step ethanol production using the combination of raw cassava starch hydrolysis and fermentation of three fermentation scales in this study is practicable and feasible for the scale-up of industrial production of ethanol from raw starch.

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