Clostridium Acetobutylicum Strain Research Articles

Mathematical modeling of ABE fermentation on glucose substrate with Zn supplementation for enhanced butanol production

Supplementation or limitation of some micronutrients during acetone-butanol-ethanol (ABE) fermentation has led to improvement in butanol yield and productivity. A mechanistic model of ABE fermentation offers insights in understanding these complex interactions and improving productivity through optimal culture conditions. This study proposes a mechanistic kinetic model of ABE fermentation by two Clostridium Acetobutylicum strains, L7 and ATCC 824 using glucose as sole carbon source without zinc and with various zinc doses. The model incorporates enzyme regulation by zinc on several glycolytic, acidogenesis and solventogenesis enzymes. The model was fitted and validated to experimental data collected from the published literature. The simulated results were in compliance with the experimental data, most importantly indicating higher glucose consumption and butanol productivity when supplemented with zinc compared to the control culture. The average squared correlation factor (R2) between the experimental and the simulated results, without and with zinc, were 0.99 and 0.96 for glucose, and 0.89 and 0.95 for butanol, respectively. A sensitivity analysis performed on the fitted and validated model indicated that the glucose consumption and growth parameters most influenced the model outputs. The developed model can be used as a template for modeling ABE fermentation under different combinations of micronutrients that may offer improved butanol yield and productivity.

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.

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.

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.

Reliable and Scalable Identification and Prioritization of Putative Cellulolytic Anaerobes With Large Genome Data.

In the era of high-throughput sequencing, genetic information that is inherently whispering hints of the microbes’ functional niches is becoming easily accessible; however, properly identifying and characterizing these genetic hints to infer the microbes’ functional niches remains a challenge. Regarding genome-centric interpretation on the specific functional niche of cellulose hydrolysis for anaerobes, often encountered in practice is a lack of confidence in predicting the anaerobes’ real cellulolytic competency based solely on abundances of the varying carbohydrate-active enzyme modules annotated or on their taxonomy affiliation. Recognition of the synergy machineries that include but not limited to the cellulosome gene clusters is equally important as the annotation of individual carbohydrate-active modules or genes. In the interpretation of complete genomes of 2,768 microbe strains whose phenotypes have been well documented, with the incorporation of an automatic recognition of synergy among the carbohydrate active elements annotated, an explicit genotype–phenotype correlation was evidenced to be feasible for cellulolytic anaerobes, and a bioinformatic pipeline was developed accordingly. This genome-centric pipeline would categorize putative cellulolytic anaerobes into six genotype groups based on differential cellulose-hydrolyzing capacity and varying synergy mechanisms. Suggested in this genotype–phenotype correlation analysis was a finer categorization of the cellulosome gene clusters: although cellulosome complexes, by their nature, could enable the assembly of a number of carbohydrate-active units, they do not certainly guarantee the formation of the cellulose–enzyme–microbe complex or the cellulose-hydrolyzing activity of the corresponding anaerobe strains, for example, the well-known Clostridium acetobutylicum strains. Also, recognized in this genotype-phenotype correlation analysis was the genetic foundation of a previously unrecognized machinery that may mediate the microbe–cellulose adhesion, to be specific, enzymes encoded by genes harboring both the surface layer homology and cellulose-binding CBM modules. Applicability of this pipeline on scalable annotation of large genome datasets was further tested with the annotation of 7,902 reference genomes downloaded from NCBI, from which 14 genomes of putative paradigm cellulose-hydrolyzing anaerobes were identified. We believe the pipeline developed in this study would be a good add as a bioinformatic tool for genome-centric interpretation of uncultivated anaerobes, specifically on their functional niche of cellulose hydrolysis.

Production of acetone, butanol, and ethanol by fermentation of Saccharina latissima: Cultivation, enzymatic hydrolysis, inhibitor removal, and fermentation

Seaweed (or macroalgae) produced sustainably at large scale opens opportunities as source of fuels, chemicals and food. The production does not directly compete with terrestrial food production and may make use of anthropogenic sources of carbon dioxide and nitrogen. Seaweed biomass can be transformed into a suitable substrate for fermentation using a biorefinery approach. In this study the entire process of biofuel production from seaweed is described: starting with cultivation and harvest, the seaweed is dried and cut, enzymatically hydrolysed, demineralized, detoxified, and finally fermented into acetone, butanol, and ethanol (ABE). Juvenile Saccharina latissima was directly seeded on AlgaeTex® nets and cultivated in the North East Atlantic off the west coast of Scotland for 6 months. Sun dried seaweed was hydrolysed with different enzymes, looking for optimal glucose release, solid/liquid ratio, and enzyme load. Using Cellic® CTec2 in combination with alginate lyases, approximately 80% of available glucose was released. The hydrolysis was scaled up to 100 L, using only Cellic® CTec2. Part of the hydrolysate was demineralized using ion-exclusion chromatography, removing over 90% of minerals while recovering 92% of glucose and mannitol. A fraction of the demineralized hydrolysate was additionally detoxified using a hydrophobic resin to remove hydrophobic components to a concentration below detection limit. The three hydrolysates (untreated, demineralized, and demineralized followed by detoxification) were used as substrate for ABE production by a newly developed strain of Clostridium acetobutylicum adapted to grow on S. latissima hydrolysate. Demineralization reduced the lag phase of fermentation from 72 h (untreated) to 24–48 h. Further detoxification of the hydrolysate led to immediate fermentation, resulting in a yield of 0.23 ± 0.02 gABE/gsugar similar to control fermentation in control medium (0.19 gABE/gsugar).

Response characteristics of the membrane integrity and physiological activities of the mutant strain Y217 under exogenous butanol stress.

Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

Simultaneous Fermentation of Mixed Sugar by a Newly Isolated Clostridium beijerinckii GSC1

A new wild type solventogenic Clostridium bacterium, which could produce a large amount of solvent from a mixture of 70% glucose and 30% xylose, was isolated. Based on 16S rRNA gene analysis, this strain was identified as Clostridium beijerinckii. Batch fermentations with this strain (C. beijerinckii GSC1) resulted in the production of 19.65 g/L total solvents (Acetone-Butanol-Ethanol), which is 31% higher than that with the typical wild type strain Clostridium acetobutylicum ATCC 824. This new strain utilized glucose and xylose simultaneously without genetic modification. The selectivity of GSC1 for butanol was 81.8%, which is much higher than that of C. acetobutylicum ATCC 824 (68.7%). Simple genetic modification was performed to obtain a more improved performance. The acid production in batch fermentation by C. beijerinckii GSC1_R1 (gene-modified strain) was reduced to 2.6 g/L from 6.02 g/L. The solvent productivity of GSC1_R1 in continuous fermentation was 3.6 g/L/h. These results indicate that the newly isolated strain is very promising and applicable for the production of biobutanol from second-generation biomass owing to the superior performance of the wild type strain.

Enhanced coproduction and trade-off of the hydrogen and butanol in the coupled system of pervaporation and repeated-cycle fixed-bed fermentation

In the study, a coupled system of pervaporation and repeated-cycle fixed bed fermentation with the bagasse as the carrier material was developed. Three repeated cycles of fermentation lasting for 540 h was efficiently carried out to coproduce hydrogen and butanol with the strain Clostridium acetobutylicum. From 1st cycle to 3rd cycle, the average hydrogen productivity and butanol productivity were around 130 mLL−1 h−1 and 0.15 gL−1 h−1, respectively. Average hydrogen yield and butanol yield were around 0.17 Lg−1 and 0.19 gg−1. The hydrogen and butanol production per unit cell weight were 27 Lg−1 and 31 gg−1, which were more than two times of those in a conventional repeated-cycle fermentation. The trade-off and competition between the hydrogen and butanol production was studied and analyzed. Compared with the pervaporation-free fermentation, the hydrogen yield was decreased by 14 % and butanol yield was increased by 27 % in the coupled system. Instantaneous solvent removal from the broth by pervaporation changed probably the metabolic pathway to more butanol production over hydrogen. From 1st cycle to 3rd cycle, the respiration heat generated during each fermentation cycle could cover 45 %, 24 % and 37 % of the total energy requirement of the pervaporation achieving the best use of the energy.

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h−1), a higher butyric acid production of 6.8 g·L−1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h−1, with a constant hydrogen production of 507 mL·h−1, corresponding to a volumetric rate of 6.10 L·L−1 d−1, and a yield of 2.39 mol of H2 per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.

Improvement of butanol production in Clostridium acetobutylicum through enhancement of NAD(P)H availability.

Clostridium acetobutylicum is a natural producer of butanol, butyrate, acetone and ethanol. The pattern of metabolites reflects the partitioning of redox equivalents between hydrogen and carbon metabolites. Here the exogenous genes of ferredoxin-NAD(P)+ oxidoreductase (FdNR) and trans-enoyl-coenzyme reductase (TER) are introduced to three different Clostridium acetobutylicum strains to investigate the distribution of redox equivalents and butanol productivity. The FdNR improves NAD(P)H availability by capturing reducing power from ferredoxin. A butanol production of 9.01g/L (36.9% higher than the control), and the highest ratios of butanol/acetate (7.02) and C4/C2 (3.17) derived metabolites were obtained in the C acetobutylicum buk- strain expressing FdNR. While the TER functions as an NAD(P)H oxidase, butanol production was decreased in the C. acetobutylicum strains containing TER. The results illustrate that metabolic flux can be significantly changed and directed into butanol or butyrate due to enhancement of NAD(P)H availability by controlling electron flow through the ferredoxin node.

A small‐scale investigation process for the Clostridium acetobutylicum production of butanol using high‐energy carbon heavy ion irradiation

Applied heavy ion irradiation technology and butanol industrial practices as a whole have been used as a strategy for the development of an attractive alternative to petroleum-based fuels. Clostridium acetobutylicum (C. acetobutylicum) strains are well documented as fermentation strains for the production of biobutanol. However, it has been reported that solvent production inhibits the growth of these strains, and the accumulation of acetate also inhibits biomass synthesis, rendering the production of butanol from acetone-butanol-ethanol (ABE) fermentation processes economically challenging. In this manuscript, we propose the use of high-energy carbon heavy ion irradiation from the Heavy Ion Research Facility in Lanzhou (HIRFL) to obtain a culture with an increased butanol yield. Our findings suggest that the use of a high-energy 12C6+ heavy ion irradiation dose of 45 Gy with an energy of 135AMeV and ion pulses/levels of 106-108 favours ABE solvent production in an irradiated strain compared with the non-irradiated strain. The strategy reported in this manuscript may contribute to the development of a cost-effective butanol fermentation process that is competitive with similar fermentation processes.

Utilization of steam-exploded corn straw to produce biofuel butanol via fermentation with a newly selected strain of Clostridium acetobutylicum

The feasibility of utilizing corn straws to produce butanol via fermentation with Clostridium acetobutylicum was evaluated. The supernatant of enzymatically hydrolyzed supernatant of steam-exploded corn straws was used as the raw material. A bacterial strain was selected from Clostridium acetobutylicum zzu-02 and Clostridium beijerinckii zzu-01, which was capable of fermenting the enzymatically hydrolyzed supernatant of steam-exploded corn straw to produce butanol with high yield. The optimal fermentation conditions for the selected strain with enzymatically hydrolyzed supernatant of steam-exploded corn straw were also investigated and they were determined as follows: sugar concentrations in enzymatically hydrolyzed solution of steam exploded corn straws, 57.5 g/L; initial pH, 6.3; the amount of added CaCO3, 5g/L; the bacterial inoculation concentration to enzymatically hydrolyzed solution, 6%; fermentation temperature, 37 oC, the amounts of the added nutritional elements, i.e. yeast extract, CH3COONH4, KH2PO4, and C6H6N2O, 0.8, 6.0, 0.5, and 0.25 g/L, respectively. Under these conditions, the butanol yield reached 9.88 g/L. Based on the butanol metabolism pathways, supplementation of a small amount of C6H6N2O was found to effectively increase the yield of butanol production.

Effects of nutritional enrichment on acid production from degenerated (non-solventogenic) Clostridium acetobutylicum strain M5

Clostridium acetobutylicum has been used as a microbial platform for the production of butanol, acetone, and butyrate from biomass. This study examined the effect of nutritional enrichment on the production of acetate and butyrate by C. acetobutylicum in culture, and tested whether this nutritional change could shift metabolic flux in these microbial cells. The degenerated (non-solventogenic) C. acetobutylicum M5 strain, which lacks the pSOL1 plasmid that contains genes responsible for solvent production, was cultured in the rich medium, C. acetobutylicum medium 1 (CAM1). As a control, M5 strain was also cultured in clostridial growth medium (CGM). Batch fermentation of M5 strain in CAM1 achieved a cell density of 23.7 (OD600), which was 2.55 times that obtained when these cells were cultured in CGM. Fermentation in CAM1 yielded volumetric acetate and butyrate productivities of 0.42 g/L/h and 1.06 g/L/h, respectively, which were 2.33 and 1.33 times the values obtained in CGM. Nutritional enrichment also increased the acetate-to-butyrate ratio, which was 0.39 g/g for M5 cells grown in CAM1 and 0.25 g/g for those grown in CGM. These findings demonstrate that the tested nutritional enrichment triggers a metabolic shift in the acid production of a degenerated C. acetobutylicum in culture.

Genomic comparison of Clostridium species with the potential of utilizing red algal biomass for biobutanol production

BackgroundSustainable biofuels, which are widely considered as an attractive alternative to fossil fuels, can be generated by utilizing various biomass from the environment. Marine biomass, such as red algal biomass, is regarded as one potential renewable substrate source for biofuels conversion due to its abundance of fermentable sugars (e.g., galactose). Previous studies focused on the enhancement of biofuels production from different Clostridium species; however, there has been limited investigation into their metabolic pathways, especially on the conversion of biofuels from galactose, via whole genomic comparison and evolutionary analysis.ResultsTwo galactose-utilizing Clostridial strains were examined and identified as Clostridium acetobutylicum strain WA and C. beijerinckii strain WB. Via the genomic sequencing of both strains, the comparison of the whole genome together with the relevant protein prediction of 33 other Clostridium species was established to reveal a clear genome profile based upon various genomic features. Among them, five representative strains, including C. beijerinckii NCIMB14988, C. diolis DSM 15410, C. pasteurianum BC1, strain WA and WB, were further discussed to demonstrate the main differences among their respective metabolic pathways, especially in their carbohydrate metabolism. The metabolic pathways involved in the generation of biofuels and other potential products (e.g., riboflavin) were also reconstructed based on the utilization of marine biomass. Finally, a batch fermentation process was performed to verify the fermentative products from strains WA and WB using 60 g/L of galactose, which is the main hydrolysate from algal biomass. It was observed that strain WA and WB could produce up to 16.98 and 12.47 g/L of biobutanol, together with 21,560 and 10,140 mL/L biohydrogen, respectively.ConclusionsThe determination of the production of various biofuels by both strains WA and WB and their genomic comparisons with other typical Clostridium species on the analysis of various metabolic pathways was presented. Through the identification of their metabolic pathways, which are involved in the conversion of galactose into various potential products, such as biobutanol, the obtained results extend the current insight into the potential capability of utilizing marine red algal biomass and provide a systematic investigation into the relationship between this genus and the generation of sustainable bioenergy.

Production of biosolvents and acids by salinity-adapted strain of clostridium acetobutylicum: Effects of salt and molasses concentrations

In this study, the growth of Clostridium acetobutylicum was evaluated in Clostridium basal medium (CBM) containing 0.001, 0.5, 1, 2 and 4 % salt concentrations. Although the strain is sensitive to salinity of more than 2 %, the adapted strain was shown to grow even at 6 % salinity. Results indicate adverse effects of salinity on bacterial growth and bioproducts, such as 1- -butanol and butyric acid, whereas the produced acetone is increased by salinity in CBM. In addition, the glucose of CBM is substituted by sugar beet molasses, due to its lower price and greater accessibility. Therefore, molasses-based mediums (MBM) at different concentrations of molasses were examined to assess the effect of molasses concentration on the adapted strain at low salinity. The results showed that 4 and 6 % concentrations of molasses are optimum concentrations for bacterial growth and its production of bioproducts at low salinity. Finally, the simultaneous effects of salinity and molasses concentration on the adapted strain were investigated. For this purpose, molasses-based mediums (MBM) containing 2, 3 and 4 % molasses concentration at 4 % salinity were considered. The results demonstrated that the increase in the molasses concentration raises the production of both butyric acid and acetone.

A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

Fed-batch fermentation with intermittent gas stripping using immobilized Clostridium acetobutylicum for biobutanol production from corn stover bagasse hydrolysate

Corn stover bagasse hydrolysate was used as the substrate for fed-batch acetone-butanol-ethanol (ABE) fermentation and in situ product removal by intermittent gas stripping. The mutant strain Clostridium acetobutylicum ABE-P 1201 was inoculated for ABE production. As results, a final cumulative butanol concentration of 18.6g/L (total ABE: 28.3g/L) was produced from 97.6g/L of total fermentable sugars (75.3g/L of glucose and 22.3g/L of xylose), with an ABE yield of 83.2g/kg of CSB. The gas stripping condensate obtained in the integrated process contained 77–136g/L of butanol and 115–220g/L of total ABE.

Enhanced butanol fermentation using metabolically engineered Clostridium acetobutylicum with ex situ recovery of butanol

In this study, metabolic target reactions for strain engineering were searched via intracellular coenzyme A (CoA) metabolite analysis. The metabolic reactions catalyzed by thiolase (AtoB) and aldehyde-alcohol dehydrogenase (AdhE1) were considered potential rate-limiting steps. In addition, CoA transferase (CtfAB) was highlighted as being important for the assimilation of organic acids, in order to achieve high butanol production. Based on this quantitative analysis, the BEKW_E1AB-atoB strain was constructed by overexpressing the thl (atoB), adhE1, and ctfAB genes in Clostridium acetobutylicum strain BEKW, which has the phosphotransacetylase (pta) and butyrate kinase (buk) genes knocked out. After 100h of continuous fermentation coupled with adsorptive ex situ butanol recovery, the concentrations found after considering desorption, yield, and productivity for the BEKW_E1AB-atoB strain were 55.7g/L, 0.38g/g, and 2.64g/L/h, respectively. The level of butanol production achieved (2.64g/L/h) represents the highest reported value obtained after adsorptive, long-term fermentation.

Production of hydrogen energy from dilute acid-hydrolyzed palm oil mill effluent in dark fermentation using an empirical model

Hydrogen generation was studied using palm oil mill effluent (POME) as an agro-industrial waste obtained from the palm oil industry. POME was subjected to a dilute acid hydrolysis step by HCl (37% v/v) to release fermentable sugars from cellulosic content. POME hydrolysate obtained was used as a substrate for hydrogen generation. The composition of POME hydrolysate showed glucose and xylose were the main monomeric sugars liberated. Hydrogen production was performed in dark fermentation process, in which the new bacterial strain Clostridium acetobutylicum YM1 was cultivated on POME hydrolysate based on a central composite design (CCD). CCD was constructed by considering three pivotal process variables including incubation temperature, initial pH of culture medium and microbial inoculum size. An empirical model, namely second-order polynomial regression model was generated and adjusted to CCD data. The analysis of empirical model generated showed that the linear and quadratic terms of temperature had a highly significant effect on hydrogen generation (P < 0.01). Furthermore, the quadratic effects of initial pH value of culture medium and inoculum size had a significant effect on hydrogen production at 95% probability level (P < 0.05). The regression model also showed that the interaction effect between temperature and initial pH value of the culture medium on the hydrogen generation was highly significant (P < 0.01). The empirical model suggested that the optimum conditions for hydrogen production were an incubation temperature of 38 °C, initial pH value of 5.85 and inoculum size of 17.61% with predicting the production of a cumulative hydrogen volume of 334.2 ml under optimum conditions. In order to validate the optimum conditions determined, C. acetobutylicum YM1 was cultivated on POME hydrolysate in optimum conditions. Verification test results showed that a cumulative hydrogen volume of 333.5 ml and a hydrogen yield of 108.35 ml H2/g total reducing sugars consumed were produced.