Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.     

The occurrence and ecology of microbial chain elongation of carboxylates in soils

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.Subject terms: Soil microbiology, Microbial ecology     

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.     

Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7T

Clostridium carboxidivorans strain P7^T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO₂ fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.Clostridium carboxidivorans strain P7^T (equivalent to ATCC BAA-624^T and DSM 15243^T) is an obligate anaerobe that can grow autotrophically with H₂ and CO₂ or CO (fixing carbon via the Wood-Ljungdahl pathway), or it can grow chemoorganotrophically with simple sugars (1). Acetate, ethanol, butanol, and butyrate are end products of metabolism.For slow-growing strict anaerobes such as Clostridium carboxidivorans, genome sequencing provides a rapid theoretical characterization of its metabolism compared to traditional methods. We isolated and amplified genomic C. carboxidivorans DNA using the Wizard genomic DNA purification kit (Promega, Madison, WI) and the REPLI-g kit (Qiagen). A single shotgun pyrosequencing run using a Genome Sequencer FLX system (454 Life Sciences, Branford, CT) resulted in 429,680 high-quality reads (mean read length, 231.6 bp) that were assembled using Newbler software (454 Life Sciences) into 225 contigs >500 bp long. Paired-end sequencing produced 111,154 reads (mean read length, 256.3 bp). Assembly of the paired-end and shotgun reads produced 73 scaffolds containing 216 large contigs with a mean sequence depth of 16.33 reads. PCR amplification and Sanger sequencing were conducted, followed by scaffold assembly using Sequencher (Gene Codes, Ann Arbor, MI). The 4.4-Mb final assembly has 33 scaffolds containing 69 contigs with a Phred-equivalent quality score of 40 or above (accuracy, >99.99%) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"ADEK00000000","term_id":"295883218","term_text":"ADEK00000000"}}ADEK00000000).The sequence was annotated using Annotation Engine (J. Craig Venter Institute) and manually curated using Manatee (http://manatee.sourceforge.net/). The genome has 29.7% G+C content and contains 4,174 protein-coding sequences, 3 rRNA operons, 1 tmRNA (dual tRNA-like and mRNA-like nature), 6 noncoding RNAs (ncRNAs), and 48 tRNA genes. (6). Comparison of 16S rRNA genes showed that C. carboxidivorans is closely related to Clostridium scatologenes ATCC 25775^T (97% sequence identity) and Clostridium drakei type strain SL1^T (99% sequence identity). C. carboxidivorans shares 94% 16S rRNA sequence identity with Clostridium ljungdahlii (4.6 Mb), another solventogenic species.Pathway analyses indicated that C. carboxidivorans is similar to other anaerobic acetogens, such as Moorella thermoacetica (8), in having an incomplete reductive tricarboxylic acid (TCA) cycle where fumarate reductase is absent. Like other acetogenic clostridia, C. carboxidivorans uses the Wood-Ljungdahl pathway for fixing carbon dioxide to organic carbon via acetyl coenzyme A (acetyl-CoA) (5). Two of these genes encode carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a complex to catalyze the carbonyl branch of the pathway for carbon fixation and acetyl-CoA production. C. carboxidivorans has genes that encode phosphotransacetylase and acetate kinase for converting acetyl-CoA into acetate, yielding ATP (2).C. carboxidivorans is unique among other known acetogenic clostridia because it can fix carbon via the Wood-Ljungdahl pathway and convert acetyl-CoA into butanol, which is more energy dense than ethanol. Both C. carboxidivorans and Clostridium acetobutylicum encode NADPH-dependent butanol dehydrogenase (74% identity) to convert acetyl-CoA into butanol (3, 4), but C. acetobutylicum cannot fix CO₂ or CO into acetyl-CoA. Conversely, C. ljungdahlii can fix CO and CO₂, but it lacks butanol dehydrogenase and cannot convert acetyl-CoA into butanol. Therefore, P7 includes beneficial properties of both these industrially important strains. The genome sequence of C. carboxidivorans P7 could potentially accelerate research allowing its industrial application for biofuel production or to enable some of its pathways to be used directly in synthetic biology for biofuel production.     

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO₂. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H⁺-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na⁺-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Effects of zinc on the production of alcohol by <Emphasis Type="Italic">Clostridium carboxidivorans</Emphasis> P7 using model syngas

Renewable energy, including biofuels such as ethanol and butanol from syngas bioconversed by Clostridium carboxidivorans P7, has been drawing extensive attention due to the fossil energy depletion and global eco-environmental issues. Effects of zinc on the growth and metabolites of C. carboxidivorans P7 were investigated with model syngas as the carbon source. The cell concentration was doubled, the ethanol content increased 3.02-fold and the butanol content increased 7.60-fold, the hexanol content increased 44.00-fold in the medium with 280 μM Zn²⁺, when comparing with those in the control medium [Zn²⁺, (7 μM)]. Studies of the genes expression involved in the carbon fixation as well as acid and alcohol production in the medium with 280 μM Zn²⁺ indicated that fdhII was up-regulated on the second day, acs A, fdhII, bdh35 and bdh50 were up-regulated on the third day and bdh35, acsB, fdhI, fdhIII, fdhIV, buk, bdh10, bdh35, bdh40 and bdh50 were up-regulated on the fourth day. The results indicated that the increased Zn²⁺ content increased the alcohol production through increase in the gene expression of the carbon fixation and alcohol dehydrogenase.     

Plant growth and responses of antioxidants of <Emphasis Type="Italic">Chenopodium album</Emphasis> to long-term NaCl and KCl stress

The effects of long-term NaCl and KCl treatment on plant growth and antioxidative responses were investigated in Chenopodium album, a salt-resistant species widely distributed in semi-arid and light-saline areas of Xinjiang, China. Growth parameters [plant height, branch number, leaf morphology and chlorophyll (Chl) content], the level of oxidative stress [superoxide anion radical (O₂ ⁻), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations], activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX)], the contents of non-enzymatic antioxidants [carotenoids (Car) and ascorbic acid (AsA)] and expression of selected genes were investigated. Plants were grown in the presence of 0, 50, and 300 mM NaCl or KCl for 2 months. Growth was stimulated by 50 mM NaCl or KCl, maintained stable at 300 mM NaCl, but was inhibited by 300 mM KCl. Three hundred mM NaCl did not affect O₂ ⁻, H₂O₂, MDA, Car and AsA, but increased the activities of SOD, CAT and POX compared to the controls. RT-PCR analysis suggested that expression of some genes encoding antioxidant enzymes could be induced during long-term salt stress, which was consistent with the enzyme activities. Treatment with 300 mM KCl was associated with elevated oxidative stress, and significantly decreased Car and AsA contents. These results suggest that an efficient antioxidant machinery is important for overcoming oxidative stress induced by treatment with high NaCl concentrations in C. album. Other strategies of ion regulation may also contribute to the differential tolerance to Na and K at higher concentrations.     

Intracellular metabolite profiling and the evaluation of metabolite extraction solvents for Clostridium carboxidivorans fermenting carbon monoxide

Clostridium carboxidivorans ferments CO, CO₂, and H₂ via the Wood-Ljungdahl pathway. CO, CO_2, and H₂ are unique substrates, unlike other carbon sources like glucose, so it is necessary to analyze intracellular metabolite profiles for gas fermentation by C. carboxidivorans for metabolic engineering. Moreover, it is necessary to optimize the metabolite extraction solvent specifically for C. carboxidivorans fermenting syngas. In comparison with glucose media, the gas media allowed significant abundance changes of 38 and 34 metabolites in the exponential and stationary phases, respectively. Especially, C. carboxidivorans cultivated in the gas media showed changes of fatty acid metabolism and higher levels of intracellular fatty acid synthesis possibly due to cofactor imbalance and slow metabolism. Meanwhile, the evaluation of extraction solvents revealed the mixture of water-isopropanol-methanol (2:2:5, v/v/v) to be the best extraction solvent, which showed a higher extraction capability and reproducibility than pure methanol, the conventional extraction solvent. This is the first metabolomic study to demonstrate the unique intracellular metabolite profiles of the gas fermentation compared to glucose fermentation, and to evaluate water-isopropanol-methanol as the optimal metabolite extraction solvent for C. carboxidivorans on gas fermentation.     

Influence of Growth Conditions on the Production of a Bacteriocin, Pediocin AcH, by Pediococcus acidilactici H

The influence of growth parameters on the production of pediocin AcH by Pediococcus acidilactici H was studied. This strain produced large quantities of pediocin AcH in TGE broth (Trypticase [1%], glucose [1%], yeast extract [1%], Tween 80 [0.2%], Mn²⁺ [0.033 mM], Mg²⁺ [0.02 mM] [pH 6.5]) within 16 to 18 h at 30 to 37°C (final pH, 3.6 to 3.7). Pediocin AcH production was negligible when the pH of the medium was maintained at 5.0 or above, even in the presence of high cell mass.     

Contribution by Saccharomyces cerevisiae yeast to fermentative flavour compounds in wines from cv. Albariño

A comparative study was made of the fermentation products of Spanish Albariño wines produced with spontaneous yeast flora and an indigenous selected Saccharomyces cerevisiae strain (Alb16). The content of fermentative volatile compounds was determined by gas-chromatography-FID. Fifteen compounds (5 alcohols, 7 esters and 3 acetates) were identified in the two Albariño wines studied. Higher alcohols, ethyl esters (except ethyl hexanoate and ethyl octanoate) and acetates were in greater concentration in the spontaneous fermentation wine than in that with selected Alb16 strain. Principal components analysis showed good separation between the different wines.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine,l-Valine,and 2-Ketoisovalerate

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (Y_P/S) of 0.36 mol per mol of glucose and a volumetric productivity (Q_P) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y_P/S of 0.24 mol per mol of glucose and a Q_P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.     

Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.