Biological hydrogen production from palm oil mill effluent (POME) by anaerobic consortia and Clostridium beijerinckii.

Palm oil mill effluent (POME) was tested as a substrate to produce hydrogen by dark fermentation. Two microbial consortia and a pure culture of Clostridium beijerinckii (ATCC 8260) were cultured anaerobically in raw, diluted and hydrolyzed POME to compare biohydrogen production yields in all three media. Experiments were done in 15 mL Hungate tubes containing 5 mL of medium and 1 mL of inoculum. When Clostridium beijerinckii was cultivated at 30 °C in the hydrolyzed POME (P003), containing 7.5 g/L of sucrose, during 8 days of fermentation and 20 % of the inoculum, the maximum biohydrogen production yield was 4.62 LH2/Lmed. Consortium C3 also showed the best production in hydrolyzed POME while consortium C6 achieved its maximum production in raw POME. This effluent is a potential substrate for biohydrogen production.

Characterization of a Clostridium beijerinckii spo0A mutant and its application for butyl butyrate production.

Spo0A is a master regulator that governs the metabolic shift of solventogenic Clostridium species such as Clostridium beijerinckii. Its disruption can thus potentially cause a significant alteration of cellular physiology as well as metabolic patterns. To investigate the specific effect of spo0A disruption in C. beijerinckii, a spo0A mutant of C. beijerinckii was characterized in this study. In a batch fermentation with pH control at 6.5, the spo0A mutant accumulated butyrate and butanol up to 8.96 g/L and 3.32 g/L, respectively from 60 g/L glucose. Noticing the unique phenotype of the spo0A mutant accumulating both butyrate and butanol at significant concentrations, we decided to use the spo0A mutant for the production of butyl butyrate that can be formed by the condensation of butyrate and butanol during the ABE fermentation in the presence of the enzyme lipase. Butyl butyrate is a value-added chemical that has numerous uses in the food and fragrance industry. Moreover, butyl butyrate as a biofuel is compatible with Jet A-1 aviation kerosene and used for biodiesel enrichment. In an initial trial of small-scale extractive batch fermentation using hexadecane as the extractant with supplementation of lipase CalB, the spo0A mutant was subjected to acid crash due to the butyrate accumulation, and thus produced only 98 mg/L butyl butyrate. To alleviate the butyrate toxicity, the biphasic medium was supplemented with 10 g/L CaCO3 and 5 g/L butanol. The butyl butyrate production was then increased up to 2.73 g/L in the hexadecane layer. When continuous agitation was performed to enhance the esterification and extraction of butyl butyrate, 3.32 g/L butyl butyrate was obtained in the hexadecane layer. In this study, we successfully demonstrated the use of the C. beijerinckii spo0A mutant for the butyl butyrate production through the simultaneous ABE fermentation, condensation, and extraction. Biotechnol. Bioeng. 2017;114: 106-112. © 2016 Wiley Periodicals, Inc.

Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO3-associated recovery of solvent production.

Degenerate Clostridium beijerinckii strain (DG-8052) can be partially recovered by supplementing CaCO3 to fermentation media. Genome resequencing of DG-8052 showed no general regulator mutated. This study focused on transcriptional analysis of DG-8052 and its response to CaCO3 treatment via microarray. The expressions of 5168 genes capturing 98.6% of C. beijerinckii NCIMB 8052 genome were examed. The results revealed that with addition of CaCO3 565 and 916 genes were significantly up-regulated, and 704 and 1044 genes significantly down-regulated at acidogenic and solventogenic phase of DG-8052, respectively. These genes are primarily responsible for glycolysis to solvent/acid production (poR, pfo), solventogensis (buk, ctf, aldh, adh, bcd) and sporulation (spo0A, sigE, sigma-70, bofA), cell motility and division (ftsA, ftsK, ftsY, ftsH, ftsE, mreB, mreC, mreD, rodA), and molecular chaperones (grpE, dnaK, dnaJ, hsp20, hsp90), etc. The functions of some altered genes in DG-8052, totalling 5.7% at acidogenisis and 8.0% at sovlentogenisis, remain unknown. The response of the degenerate strain to CaCO3 was suggested significantly pleiotropic. This study reveals the multitude of regulatory function that CaCO3 has in clostridia and provides detailed insights into degeneration mechanisms at gene regulation level. It also enables us to develop effective strategies to prevent strain degeneration in future.

Proteomic analysis to elucidate degeneration of Clostridium beijerinckii NCIMB 8052 and role of Ca(2+) in strain recovery from degeneration.

Degeneration of solventogenic Clostridium strains is one of the major barriers in bio-butanol production. A degenerated Clostridium beijerinckii NCIMB 8052 strain (DG-8052) was obtained without any genetic manipulation. Supplementation of CaCO3 to fermentation medium could partially recover metabolism of DG-8052 by more than 50 % increase of cell growth and solvent production. This study investigated the protein expression profile of DG-8052 and its response to CaCO3 treatment. Compared with WT-8052, the lower expressed proteins were responsible for disruption of RNA secondary structures and DNA repair, sporulation, signal transduction, transcription regulation, and membrane transport in DG-8052. Interestingly, accompanied with the decreased glucose utilization and lower solvent production, there was a decreased level of sigma-54 modulation protein which may indicate that the level of sigma-54 activity may be associated with the observed strain degeneration. For the addition of CaCO3, proteomic and biochemical study results revealed that besides buffer capacity, Ca(2+) could stabilize heat shock proteins, increase DNA synthesis and replication, and enhance expression of solventogenic enzymes in DG-8052, which has a similar contribution in WT-8052.

Maintenance of DeltapH by a butanol-tolerant mutant of Clostridium beijerinckii.

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane DeltapH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The DeltapH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the DeltapH component of the protonmotive force) and supplemented with glucose and Mg2+. The DeltapH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a DeltapH of 1.4 when butanol was added to a concentration of 1.5 % (w/v), while the wild-type DeltapH was reduced to 0.1. The DeltapH of both strains was completely dissipated with 1.75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the DeltapH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0.8 %) prevented this DeltapH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in DeltapH occurred more slowly than in the mutant, and sparing of the DeltapH by 0.8 % butanol was less pronounced. The authors interpret these data to mean that the DeltapH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.