Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii

An examination of the replication origin and stability determinant associated with the CAK1 filamentous viruslike particle recovered from Clostridium beijerinckii NCIMB 6444 was carried out. Seven deletion derivatives, pCKE, pCEP1, pDT5, pCKP, pDTH102, pYL102E and pYL102, were constructed and transformed into C. beijerinckii NCIMB 8052. The successful transformation of pCKE, pDT5, pCKP, pDTH102, pYL102E and pYL102 into C. beijerinckii 8052, together with the corresponding recovery of single-stranded DNA from Escherichia coli indicated that the double- and single-stranded replication origins are located on a 0.4-kb CAK1 DNA fragment. Sequence analysis of the putative 0.4-kb replication origin region of CAK1 reveals a nick site containing 22 base pairs that has homology with plasmids pC194 and pUB110 and suggests the presence of a 2.0-kb DNA region involved in stability. The putative Rep protein of CAK1 contains three conserved motifs and three essential residues of the catalytic site in agreement with Rep proteins associated with the pC194 family. The utility of the developed CAK1-derived phagemid designated pYL102E was evaluated by using it to examine heterologous expression of: (1) the manA gene derived from Thermoanaerobacterium polysaccharolyticum in E. coli and C. beijerinckii NCIMB 8052 and (2) the sol operon derived from Clostridium acetobutylicum DSM 792 in C. beijerinckii SA-2. Journal of Industrial Microbiology & Biotechnology (2002) 28, 118–126 DOI: 10.1038/sj/jim/7000225 Received 12 September 2000/ Accepted in revised form 23 October 2001     

Elucidating and alleviating impacts of lignocellulose-derived microbial inhibitors on Clostridium beijerinckii during fermentation of Miscanthus giganteus to butanol

Fermentation of liquid hot water (LHW) pretreated Miscanthus giganteus (MG) by Clostridium beijerinckii NCIMB 8052 was investigated towards understanding the toxicity of lignocellulose-derived inhibitors to solventogenic Clostridium species vis-à-vis butanol production. While C. beijerinckii NCIMB 8052 did not grow in undiluted MG hydrolysate-based fermentation medium, supplementation of this medium with Calcium carbonate enabled the growth of C. beijerinckii NCIMB 8052 and production of butanol. Using high-performance liquid chromatography (HPLC) and spectrophotometric assays, LHW-pretreated MG was found to contain lignocellulose-derived microbial inhibitory compounds; some of which were transformed by exponentially growing C. beijerinckii to less inhibitory compounds during fermentation. Contrary to all expectations, the reduction product of furfural, furfuryl alcohol, inhibited butanol production by C. beijerinckii by more than 16 %. Collectively, these results provide new insights into why lignocellulosic biomass hydrolysates are recalcitrant to fermentation to biofuels and chemicals.     

Development of a cost-effective glucose-corn steep medium for production of butanol by Clostridium beijerinckii

Corn steep water (CSW) medium (1.6% solids plus 6% glucose) was evaluated for growth and butanol production by Clostridium beijerinckii NCIMB 8052 wild-type and hyper-amylolytic, hyper-butanol-producing mutant strain BA101. CSW alone was not a suitable substrate, whereas addition of glucose supported growth and butanol production by both strains. In a batch-scale fermentation using an optimized 6% glucose-1.6% solids CSW medium, C. beijerinckii NCIMB 8052 and strain BA101 produced 10.7 g L⁻¹ and 14.5 g L⁻¹ of butanol, respectively. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and strain BA101 were 14 g L⁻¹ and 20 g L⁻¹, respectively. Initial fermentation in small-scale flasks containing 6% maltodextrin-1.6% solids concentration CSW medium resulted in 6 g L⁻¹ and 12.6 g L⁻¹ of butanol production by C. beijerinckii NCIMB 8052 and strain BA101, respectively. CSW can serve as an economic source of nitrogen, vitamins, amino acids, minerals, and other nutrients. Thus, it is feasible to use 6% glucose-1.6% solids CSW medium in place of semi-defined P2 medium. Received 9 February 1998/ Accepted in revised form 1 September 1998     

Examination of Physiological and Molecular Factors Involved in Enhanced Solvent Production by Clostridium beijerinckii BA101

The specific activities and the mRNA expression levels associated with coenzyme A transferase, acetoacetate decarboxylase, and butyraldehyde dehydrogenase were elevated in hyper-solvent-producing Clostridium beijerinckii BA101 during the exponential growth phase. The increase in expression of the sol operon and associated enzyme activities may be responsible for enhanced solvent production by C. beijerinckii BA101.     

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

Strain degeneration in solventogenic clostridia is a known problem in the technical acetone–butanol fermentation bioprocess, especially in the continuous process mode. Clostridial strain degeneration was studied by Fourier transform infrared (FT-IR) spectroscopy of the bacterial cells. Degenerative variant formation in two strains, Clostridium beijerinckii NCIMB 8052 and Clostridium species AA332, was detected spectroscopically. Colonies on solid media were sampled, or assayed directly in situ by IR microscopy. It has previously been shown that the distinctive acidogenic and solventogenic physiological phases of Clostridium acetobutylicum in liquid medium can be discriminated by FT-IR spectroscopy. This was confirmed here for C. beijerinckii NCIMB 8052. The proportion of degenerate cells in a mixed population in liquid medium could be quantified, as the spectral features change in different ways during the normal growth cycle of wild type organisms and degenerate variants in batch culture. This opens a new perspective for physiology-based process monitoring and control, especially of the continuous acetone–butanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 314–321. Received 06 October 2000/ Accepted in revised form 20 April 2001     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation

Lignocellulose-derived microbial inhibitors such as furfural and 5-hydroxymethyl furfural adversely affect fermentation of lignocellulosic biomass hydrolysates to fuels and chemicals due to their toxicity on fermenting microbes. To harness the potential of lignocellulose as a cheap source of fermentable sugars, in situ detoxification of furfural and other lignocellulose-derived microbial inhibitors is essential. To enhance in situ detoxification and tolerance of furfural by Clostridium beijerinckii NCIMB 8052 during acetone-butanol-ethanol (ABE) fermentation, the effect of glycerol on NADH/NADPH generation and ABE production by furfural (4, 5, and 6 g/L)-challenged cultures was investigated in this study. In all instances, beneficial outcomes were observed. For example, the fermentation medium supplemented with glycerol and subjected to 5 g/L furfural elicited up to 1.8- and 3-fold increases, respectively, in NADH and NADPH levels in C. beijerinckii 8052 relative to the control culture. These critical changes are the likely underpinnings for the glycerol-mediated 2.3-fold increase in the rate of detoxification of 5 g/L furfural, substrate consumption, and ABE production compared to the unsupplemented medium. Collectively, these results demonstrate that increased intracellular NADH/NADPH in C. beijerinckii 8052 due to glycerol utilization engenders favorable effects on many aspects of cellular metabolism, including enhanced furfural reduction and increased ABE production.     

Clostridium beijerinckii Cells Expressing Neocallimastix patriciarum Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.     

Glucose Uptake in Clostridium beijerinckii NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101

Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyperproducing mutant, were examined during various stages of growth. Glucose uptake in C. beijerinckii BA101 was repressed 20% by 2-deoxyglucose and 25% by mannose, while glucose uptake in C. beijerinckii 8052 was repressed 52 and 28% by these sugars, respectively. We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts of C. beijerinckii BA101 by glucose phosphorylation by PEP. The PTS activity associated with C. beijerinckii BA101 was 50% of that observed for C. beijerinckii 8052. C. beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol. Glucose phosphorylation by cell extracts derived from both C. beijerinckii BA101 and 8052 was also dependent on the presence of ATP, a finding consistent with the presence of glucokinase activity in C. beijerinckii extracts. ATP-dependent glucose phosphorylation was predominant during the solventogenic stage, when PEP-dependent glucose phosphorylation was dramatically repressed. A nearly twofold-greater ATP-dependent phosphorylation rate was observed for solventogenic stage C. beijerinckii BA101 than for solventogenic stage C. beijerinckii 8052. These results suggest that C. beijerinckii BA101 is defective in PTS activity and that C. beijerinckii BA101 compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Use of Proteomic Analysis To Elucidate the Role of Calcium in Acetone-Butanol-Ethanol Fermentation by Clostridium beijerinckii NCIMB 8052

Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO₃. We observed increases in the levels of different heat shock proteins (GrpE and DnaK), sugar transporters, and proteins involved in DNA synthesis, repair, recombination, and replication. We also noted significant decreases in the levels of proteins involved in metabolism, nucleic acid stabilization, sporulation, oxidative and antibiotic stress responses, and signal transduction. We determined that CaCO₃ enhances ABE fermentation due to both its buffering effects and its ability to influence key cellular processes, such as sugar transport, butanol tolerance, and solventogenesis. Moreover, activity assays in vitro for select solventogenic enzymes revealed that part of the underpinning for the CaCO₃-mediated increase in the level of ABE fermentation stems from the enhanced activity of these catalysts in the presence of Ca²⁺. Collectively, these proteomic and biochemical studies provide new insights into the multifactorial basis for the stimulation of ABE fermentation and butanol tolerance in the presence of CaCO₃.     

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Evidence for the Presence of an Alternative Glucose Transport System in Clostridium beijerinckii NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.     

The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli

The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit K_mS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase.     

<Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> mutant with high inhibitor tolerance obtained by low-energy ion implantation

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l⁻¹ of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g⁻¹. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l⁻¹ of ABE with a yield of 0.34 g g⁻¹, including 2.2 g l⁻¹ acetone, 6.8 g l⁻¹ butanol, and 0.5 g l⁻¹ ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.     

Truncation of Peptide Deformylase Reduces the Growth Rate and Stabilizes Solvent Production in Clostridium beijerinckii NCIMB 8052

The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn1545 mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the fms (def) gene, which encodes peptide deformylase (PDF). The C. beijerinckii fms gene product contains four diagnostic residues involved in the Zn²⁺ coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from Escherichia coli and Thermus thermophilus, the C. beijerinckii PDF can apparently tolerate N-terminal truncation. The Tn1545 insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retains at least partial function, and it complements an fms(Ts) strain of E. coli. Northern hybridizations indicate that the mutant gene is actively transcribed in C. beijerinckii. This can only occur from a previously unsuspected, outwardly directed promoter located close to the right end of Tn1545. The Tn1545 insertion in fms causes a reduction in the growth rate of C. beijerinckii, and, associated with this, the bacteria display an enhanced stability of solvent production. The latter phenotype can be mimicked in the wild type by reducing the growth rate. Therefore, the observed amelioration of degeneration in the mutants is probably due to their reduced growth rates.