Clostridium Acetobutylicum Research Articles

Performance of clostridium species and autochthonous bacteria from citrus wastewater under different carbon sources to produce biofuels

This study evaluated the energy recovery of citrus wastes with pure cultures (Clostridium acetobutylicum ATCC 824, Clostridium beijerinckii ATCC 10132 – Inocula 1 and 2, respectively) and an autochthonous microbial (Inoculum 3) from citrus wastewater (CW) in anaerobic batch reactors to produce biofuels (H2 and ethanol) fed with different carbon sources (glucose, maltose, fructose, sucrose, xylose, lactose, starch, acetate, and glycerol) in addition to CW. 4 conditions are tested: 1st - Inoculum 3, composed by Bacillus (20.44%), Clostridium (13.65%) and Lactobacillus (10.61%) was able to produce H2 with sucrose, glucose and fructose. 2nd - The highest metabolites were produced with TYA medium with Inocula 1 (374 mg L−1 of acetic acid) and 2 (611 mg L−1 of butanol). Inoculum 3 generated the highest ethanol (1571.8 mg L−1) in PYG medium. 3rd - In the same nutritional conditions (PYG), Inocula 1 and 2 produced H2 (36.80 and 64.10 mmol L−1, respectively) and acetic acid (246 and 201 mg L−1, respectively); the H2 was suppressed with inoculum 3 due to Lactobacillus genus. 4th H2 production was favored by autochthonous bacteria in CW. This study valued the autochthonous microorganisms in the possibility of bioconversion of organic matter into value-added products in addition to contribute to an environment-sustainable solution for citrus processing.

Total Synthesis of Clostrienose.

This paper considers the total synthesis of a cellular differentiation regulator of Clostridium acetobutylicum, clostrienose, which is a unique fatty-acid glycosyl ester consisting of clostrienoic acid, (3R,5E,8E,10E)-3-hydroxy-tetradeca-5,8,10-trienoic acid and α-d-galactofuranosyl-(1 → 2)-α-l-rhamnose. The key features of our synthesis include stereoselective construction of a skipped-triene system in clostrienoic acid and its esterification with a disaccharide residue. The partially protected clostrienoic acid employed for the coupling also served for the preparation of l-rhamnosyl clostrienoate, thus leading to confirmation of the proposed structure unambiguously.

Biohydrogen Gas/Acetone-Butanol-Ethanol Production from Agave Guishe Juice as a Low-Cost Growing Medium

Different strategies have been assessed for the revalorization of guishe to obtain biomolecules. The juice obtained after the mechanical extraction of guishe is rich in phytochemicals and sugars which can be converted to other products. The objective of the present study was to evaluate the production of hydrogen and butanol at different guishe juice concentrations (and therefore, different sugar concentrations) via fermentation in batch mode using Clostridium acetobutylicum ATCC 824. Fermentation assays were performed in triplicate under anaerobic conditions at 35 °C for 142 h. Guishe juice was supplemented with all components of synthetic medium (salts, vitamins and reducing agents), except glucose, and diluted at different concentrations: 20%, 40%, 60%, 80% and 100%. For comparison purposes, a control was carried out in a synthetic medium using glucose as carbon source. Results showed a maximum butanol concentration of 5.39 g/L using 80% guishe juice, corresponding to a productivity and yield of 0.04 g/L h−1 and 0.24 g/g, respectively. Meanwhile, the highest productivity (1.16 L H2/L d−1; 1.99 mmol H2/L h−1) and yield (18.4 L/kg) of hydrogen were obtained with 40% guishe juice. This study demonstrates the potential of guishe juice to be used as a low-cost substrate for hydrogen and butanol production.

Utilization of lignocellulosic biomass by glycerol organosolv pretreatment for biobutanol production integrated with bioconversion of residual glycerol into value-added products

Glycerol organosolv pretreatment (GOP) is considered an efficient method to deconstruct lignocellulose for producing fermentable sugars. Herein, the liquid fraction containing glycerol after GOP was utilized for recycled pretreatment of corn stover (CS) for four cycles. Enzymatic yield of glucose after recycled pretreatment was enhanced by 2.4–3.5 folds compared with untreated CS. Meanwhile, residual glycerol was used as carbon source for cultivation of Pichia pastoris to obtain high cell-density, and a final titer of 1.3 g/L human lysozyme was produced by P. pastoris under low temperature methanol induction strategy. Additionally, the pretreated CS was mixed with cassava as fermentable substrates for butanol production by wild-type Clostridium acetobutylicum ATCC 824. Final butanol production of 13.9 g/L was obtained from mixed substrates (25%:75% of CS/cassava) at 10% solids loading by simultaneous saccharification and fermentation. Overall, integration of residual glycerol utilization and butanol production by microbial fermentation provided an efficient strategy for biorefinery.

Municipal green waste as substrate for the microbial production of platform chemicals

In Germany alone, more than 5·106 tons of municipal green waste is produced each year. So far, this material is not used in an economically worthwhile way. In this work, grass clippings and tree pruning as examples of municipal green waste were utilized as feedstock for the microbial production of platform chemicals. A pretreatment procedure depending on the moisture and lignin content of the biomass was developed. The suitability of grass press juice and enzymatic hydrolysate of lignocellulosic biomass pretreated with an organosolv process as fermentation medium or medium supplement for the cultivation of Saccharomyces cerevisiae, Lactobacillus delbrueckii subsp. lactis, Ustilago maydis, and Clostridium acetobutylicum was demonstrated. Product concentrations of 9.4 gethanol L−1, 16.9 glactic acid L−1, 20.0 gitaconic acid L−1, and 15.5 gsolvents L−1 were achieved in the different processes. Yields were in the same range as or higher than those of reference processes grown in established standard media. By reducing the waste arising in cities and using municipal green waste as feedstock to produce platform chemicals, this work contributes to the UN sustainability goals and supports the transition toward a circular bioeconomy.Graphical

A Novel: Low-cost Method for the Isolation of Anaerobic Hydrogen Producing Bacteria

The hydrogen content of the two sludge samples namely TDS and PLS were increased after the heat shock pretreatment from 239.23 ± 10.23 mL/L to 663.87 ± 15.99 mL/L at 80°C and from 173.61 ± 17.21 mL/L to 496.74 ± 30.21 mL/L at 60°C, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) indicated that Enterococcus and Clostridium were the dominant bacteria in the TDS and PLS consortium. Two strains were successfully isolated from TDS and PLS using a simple method. Based on Bergey’s key identification and 16S rDNA sequencing, they were identified as Clostridium butyricum strain TD and Clostridium acetobutylicum strain PL. The hydrogen content, glucose consumed yield, and hydrogen yield of C. butyricum TD and C. acetobutylicum PL were 545.24 ± 7.31 mL/L, 90 ± 0.41 %, 0.89 ± 0.01 mol H2/mol glucose, and 729.34 ± 22.81 mL/L, 92.77 ± 1.39 %, 1.26 ± 0.05 mol H2/mol glucose, respectively.

Exploring the Influence of pH on the Dynamics of Acetone-Butanol-Ethanol Fermentation.

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10mM) resulted in the production of 4.4g/L isopropanol and 19.8g/L Isopropanol-Butanol-Ethanol mixture.

The Lanthipeptide Synthetase-like Protein CA_C0082 Is an Effector of Agr Quorum Sensing in Clostridium acetobutylicum.

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described.

Differential Binding of NLRP3 to non-oxidized and Ox-mtDNA mediates NLRP3 Inflammasome Activation

The NLRP3 inflammasome is a key mediator of the innate immune response to sterile tissue injury and is involved in many chronic and acute diseases. Physically and chemically diverse agents activate the NLRP3 inflammasome. Here, we show that NLRP3 binds non-oxidized and Ox-mtDNA differentially, with a half maximum inhibitory concentration (IC50) for non-oxidized and Ox-mtDNA of 4 nM and 247.2 nM, respectively. The NLRP3 Neonatal-Onset Multisystem Inflammatory Disease (NOMID) gain of function mutant could bind non-oxidized mtDNA but had higher affinity for Ox-mtDNA compared to WT with an IC50 of 8.1 nM. NLRP3 lacking the pyrin domain can bind both oxidized and non-oxidized mtDNA. Isolated pyrin domain prefers Ox-mtDNA. The NLRP3 pyrin domain shares a protein fold with DNA glycosylases and generate a model for DNA binding based on the structure and sequence alignment to Clostridium acetobutylicum and human OGG1, an inhibitor of Ox-mtDNA generation, 8-oxoguanine DNA glycosylases. We provide a new model for how NLRP3 interacts with Ox-mtDNA supported by DNA binding in the presence of a monoclonal antibody against the pyrin domain. These results give new insights into the mechanism of inflammasome assembly, and into the function of reactive oxygen species in establishing a robust immune response.

Enhancement of biohydrogen production in Clostridium acetobutylicum ATCC 824 by overexpression of glyceraldehyde-3-phosphate dehydrogenase gene

In the dark fermentation of hydrogen, development of production host is crucial as bacteria act on substrates and produce hydrogen. The present study aimed to improve hydrogen production through the development of Clostridium acetobutylicum as a superior biohydrogen producer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which produces NADH/NADPH for metabolites and energy in primary pathways, was introduced to enhance hydrogen production. The strain CAC824-G containing gapC that encodes GAPDH showed a 66.3 % higher hydrogen production than the wild-type strain, with increased NADH and NADPH pools. Glucose consumption and other byproducts, such as acetone, butanol, and ethanol, were also high in CAC824-G. Overexpression of gapC resulted in increased hydrogen production with sugars obtained from different biomass, even in the presence of inhibitors such as vanillin, 5-hydroxymethylfufural, acetic acid, and formic acid. Our results imply that overexpression of gapC in Clostridium is possible to expand the production of the reported biochemicals to produce hydrogen.

A scale-up study of the continuous ABE fermentation in a packed bed coupled with the extraction/gas-stripping in situ butanol recovery process

A 5-L pilot-scale packed-bed bioreactor with integrated in situ extraction/gas-stripping was designed and manufactured for automated acetone-butanol-ethanol (ABE) fermentation using immobilized Clostridium acetobutylicum. A continuous run of 770-h demonstrated the robustness of the bioreactor. Optimal inoculation protocols were developed to prevent mass transfer hindrance and improve butanol productivity and bacterial growth. At a dilution rate of 0.1 h−1, an improvement of 0.44 g L−1 h−1 in butanol productivity was observed with the continuous process coupled with extraction/gas-stripping. However, it was found that the bacterial formation within the packed bed was inhomogeneous, with dense bacterial growth observed only at the bottom. Therefore, inhomogeneous bacterial growth cannot be simply overcome with the in situ extraction/gas-stripping process. This highlights the need to address the issue of inhomogeneous bacterial growth in pilot-scale packed-bed reactors to improve their efficiency.

Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables a Significant Increase in Citric Acid Production

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

Heterologous 1,3-Propanediol Production Using Different Recombinant Clostridium beijerinckii DSM 6423 Strains.

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_Ppta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach.

Antisense-acting riboswitches: A poorly characterized yet important model of transcriptional regulation in prokaryotic organisms.

Riboswitches are RNA elements involved in regulating genes that participate in the biosynthesis or transport of essential metabolites. They are characterized by their ability to recognize their target molecules with high affinity and specificity. Riboswitches are commonly cotranscribed with their target genes and are located at the 5' end of their transcriptional units. To date, only two exceptional cases of riboswitches being situated at the 3' end and transcribing in the antisense direction of their regulated genes have been described. The first case involves a SAM riboswitch located at the 3' end of the ubiG-mccB-mccA operon in Clostridium acetobutylicum involved in converting methionine to cysteine. The second case concerns a Cobalamin riboswitch in Listeria monocytogenes that regulates the transcription factor PocR related to this organism's pathogenic process. In almost a decade since the first descriptions of antisense-acting riboswitches, no new examples have been described. In this work, we performed a computational analysis to identify new examples of antisense-acting riboswitches. We found 292 cases in which, according to the available information, we infer that the expected regulation of the riboswitch is consistent with the signaling molecule it senses and the metabolic function of the regulated gene. The metabolic implications of this novel type of regulation are thoroughly discussed.

Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways

BackgroundDeveloping new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO2, and H2, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands.ResultsThis work reports the development and optimization of an electrotransformation protocol for A. wieringae strain JM, which can also be used in A. wieringae DSM 1911, and A. woodii DSM 1030. We also show the functionality of the thiamphenicol resistance marker, catP, and the functionality of the origins of replication pBP1, pCB102, pCD6, and pIM13 in all tested Acetobacterium strains, with transformation efficiencies of up to 2.0 × 103 CFU/μgDNA. Key factors affecting electrotransformation efficiency include OD600 of cell harvesting, pH of resuspension buffer, the field strength of the electric pulse, and plasmid amount. Using this method, the acetone production operon from Clostridium acetobutylicum was efficiently introduced in all tested Acetobacterium spp., leading to non-native biochemical acetone production via plasmid-based expression.ConclusionsA. wieringae can be electrotransformed at high efficiency using different plasmids with different replication origins. The electrotransformation procedure and tools reported here unlock the genetic and metabolic manipulation of the biotechnologically relevant A.wieringae strains. For the first time, non-native acetone production is shown in A. wieringae.

Alleviation of Carbon Catabolite Repression through araR and xylR Inactivation in Clostridium acetobutylicum DSM 792.

Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of xylR (CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of araR (CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both araR and xylR genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both araR and xylR in C. acetobutylicum. As suggested by these data, overexpression of xylA and xylB led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars. IMPORTANCE C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them.

Construction of a Microbial Consortium for the De Novo Synthesis of Butyl Butyrate from Renewable Resources.

Butyl butyrate has shown wide applications in food, cosmetic, and biofuel sectors. Currently, biosynthesis of butyl butyrate still requires exogenous addition of precursors and lipase, which increases the production cost and limits further large-scale development. In this study, a microbial consortium was first designed to realize direct butyl butyrate production from lignocellulose. The highest butyl butyrate concentration of 34.42 g/L was detected in the solvent phase from 60 g/L glucose using a microbial coculture system composed of Clostridium acetobutylicum NJ4 and Clostridium tyrobutyricum LD with the elimination of butyric acid supplementation. Meanwhile, 13.52 g/L butyl butyrate was synthesized from 60 g/L glucose using a microbial consortium composed of three strains including strain NJ4, strain LD, and Escherichia coli BL21- pET-29a(+)-LE without the addition of any exogenous precursors and lipase. In addition, 2.94 g/L butyl butyrate could be directly produced from 60 g/L microcrystalline cellulose when Trichoderma asperellum was added to the above-mentioned three-strain microbial consortium. This four-strain microbial consortium represents the first study regarding the direct butyl butyrate production from lignocellulose without the supplementation of exogenous precursors and lipase, which may be extended to the biosynthesis of other short-chain esters, such as ethyl acetate and butyl lactate.

Confirmation of Glucose Transporters through Targeted Mutagenesis and Transcriptional Analysis in Clostridium acetobutylicum

The solvent-producing bacterium Clostridium acetobutylicum is able to grow on a variety of carbohydrates. The main hexose transport system is the phosphoenolpyruvate-dependent phosphotransferase system (PTS). When the gene glcG that encodes the glucose transporter was inactivated, the resulting mutant glcG::int(1224) grew as well as the wild type, yet its glucose consumption was reduced by 17% in a batch fermentation. Transcriptomics analysis of the phosphate-limited continuous cultures showed that the cellobiose transporter GlcCE was highly up-regulated in the mutant glcG::int(1224). The glcCE mutation did not affect growth and even consumed slightly more glucose during solventogenesis growth compared to wild type, indicating that GlcG is the primary glucose-specific PTS. Poor growth of the double mutant glcG::int(1224)-glcCE::int(193) further revealed that GlcCE was the secondary glucose PTS and that there must be other PTSs capable of glucose uptake. The observations obtained in this study provided a promising foundation to understand glucose transport in C. acetobutylicum.