Carboxydotrophic Bacteria Research Articles

Microbial co-cultivation: Reaction dynamics of combustible gases bioremediation in goaf

The accumulation of combustible gases in goaf is a key and difficult point in mine disaster prevention and control, which can easily lead to coal mine accidents and seriously safety production. Methanotrophs (S2, T7) and carboxydotrophic bacteria (M13) were extracted from mine environment to cocultivation. The cultivation conditions such as inoculation, temperature, and pH were selected to explore the effects of the growth and metabolic characteristics, and reveal the reaction kinetics of mixed strains (S2/M13, T7/M13). The results showed that the optimal inoculation amount for mixed strains was 5 %, and the maximum range of mixed carbon source inoculation for S2/M13 and T7/M13 were 9.69 % CH4+(7.27–9.69 % CO) and 9.69 % CH4+(4.35–7.27 % CO), the optimal cultivation temperatures were 25, 30 ℃, respectively, and the optimal pH was 7. In addition, the CH4 metabolic rates of S2/M13 and T7/M13 were 72.4 % and 85.4 %, respectively, and the CO metabolic rate reached over 95 % under the above optimal conditions, the processes of growth and metabolism of the mix cultures remained consistent. Moreover, the growth curves of the S2/M13 and T7/M13 conformed to the Logistic model, and whose attenuation coefficients (b) were 0.035, −0.215 d−1, the maximum specific growth rates (umax) were 0.524, 0.862 d−1, respectively. At the same time, the reaction rate of combustible gases showed high consistency in the logarithmic and stationary phases of the mixed strains, and exhibited a good linear relationship with the consumption rate of O2, the inhibitory kinetics of mixed strains on the reaction rate of combustible gases under different cultivation conditions were determined. The research results have vital theoretical and practical significance for the prevention and control of combustible gases and the safe mining in coal mines.

Bioconversion of C1 gases and genetic engineering modification of gas-utilizing microorganisms

C1 gases including CO, CO2 and CH4, are mainly derived from terrestrial biological activities, industrial waste gas and gasification syngas. Particularly, CO2 and CH4 are two of the most important greenhouse gases contributing to climate change. Bioconversion of C1 gases is not only a promising solution to addressing the problem of waste gases emission, but also a novel route to produce fuels or chemicals. In the past few years, C1-gas-utilizing microorganisms have drawn much attention and a variety of gene-editing technologies have been applied to improve their product yields or to expand product portfolios. This article reviewed the biological characteristics, aerobic or anaerobic metabolic pathways as well as the metabolic products of methanotrophs, autotrophic acetogens, and carboxydotrophic bacteria. In addition, gene-editing technologies (e.g. gene interruption technology using homologous recombination, group Ⅱ intron ClosTron technology, CRISPR/Cas gene editing and phage recombinase-mediated efficient integration of large DNA fragments) and their application in these C1-gas-utilizing microorganisms were also summarized.

Bio‑hydrogen and VFA production from steel mill gases using pure and mixed bacterial cultures

A major source of CO2 emissions is the flaring of steel mill gas. This work demonstrated the enrichment of carboxydotrophic bacteria for converting steel mill gas into volatile fatty acids and H2, via gas fermentation. Several combinations of pure and mixed anaerobic cultures were used as inoculum in 0.5-L reactors, operated at 30 and 60 °C. The process was then scaled up in a 4-L membrane bioreactor, operated for 20 days, at 48 °C. The results showed that the enriched microbiomes can oxidize CO completely to produce H2/H+ which is subsequently used to fix the CO2. At 30 °C, a mixture of acetate, isobutyrate and propionate was obtained while H2 and acetate were the main products at 60 °C. The highest CO conversion and H2 production rate observed in the membrane bioreactor were 29 and 28 mL/LR/h, respectively. The taxonomic diversity of the bacterial community increased and the dominant species was Pseudomonas.

Exploiting Aerobic Carboxydotrophic Bacteria for Industrial Biotechnology.

Aerobic carboxydotrophic bacteria are a group of microorganisms which possess the unique trait to oxidize carbon monoxide (CO) as sole energy source with molecular oxygen (O2) to produce carbon dioxide (CO2) which subsequently is used for biomass formation via the Calvin-Benson-Bassham cycle. Moreover, most carboxydotrophs are also able to oxidize hydrogen (H2) with hydrogenases to drive the reduction of carbon dioxide in the absence of CO. As several abundant industrial off-gases contain significant amounts of CO, CO2, H2 as well as O2, these bacteria come into focus for industrial application to produce chemicals and fuels from such gases in gas fermentation approaches. Since the group of carboxydotrophic bacteria is rather unknown and not very well investigated, we will provide an overview about their lifestyle and the underlying metabolic characteristics, introduce promising members for industrial application, and give an overview of available genetic engineering tools. We will point to limitations and discuss challenges, which have to be overcome to apply metabolic engineering approaches and to utilize aerobic carboxydotrophs in the industrial environment.

Specific detection of "Clostridium autoethanogenum", Clostridium ljungdahlii and Clostridium carboxidivorans in complex bioreactor samples.

The high genetic similarity between some carboxydotrophic bacteria does not allow for the use of common sequencing techniques targeting the 16S rRNA gene for species identification. 16S rRNA sequencing fails to discriminate among Clostridium ljungdahlii and 'Clostridium autoethanogenum', despite this two species exhibit significant differences in CO2 assimilation and alcohol production. In this work we designed PCR primers targeting for the DNA gyrase subunit A (gyrA) and a putative formate/nitrite transporter (fnt) to specifically detect the presence of 'C. autoethanogenum', C. ljungdahlii or Clostridium carboxidivorans. We could confirm the simultaneous presence of C. ljungdahlii and 'C. autoethanogenum' in different bioreactors, and a preference of the latter for high CO2 content.

Enrichment of syngas-converting mixed microbial consortia for ethanol production and thermodynamics-based design of enrichment strategies

BackgroundThe production of ethanol through the biochemical conversion of syngas, a mixture of H2, CO and CO2, has been typically studied using pure cultures. However, mixed microbial consortia may offer a series of benefits such as higher resilience and adaptive capacity, and non-sterile operation, all of which contribute to reducing the utility consumption when compared to pure culture-based processes. This work focuses on the study of strategies for the enrichment of mixed microbial consortia with high ethanologenic potential, investigating the effect of the operational conditions (pH and yeast extract addition) on both the ethanol yield and evolution of the microbial community along the enrichment process. The pH was selected as the main driver of the enrichment as it was expected to be a crucial parameter for the selection of carboxydotrophic bacteria with high ethanologenic potential. Additionally, a thermodynamic analysis of the network of biochemical reactions carried out by syngas-converting microbial consortia was performed and the potential of using thermodynamics as a basis for the selection of operational parameters favoring a specific microbial activity was evaluated.ResultsAll enriched consortia were dominated by the genus Clostridium with variable microbial diversity and species composition as a function of the enrichment conditions. The ethanologenic potential of the enriched consortia was observed to increase as the initial pH was lowered, achieving an ethanol yield of 59.2 ± 0.2% of the theoretical maximum in the enrichment at pH 5. On the other hand, yeast extract addition did not affect the ethanol yield, but triggered the production of medium-chain fatty acids and alcohols. The thermodynamic analysis of the occurring biochemical reactions allowed a qualitative prediction of the activity of microbial consortia, thus enabling a more rational design of the enrichment strategies targeting specific activities. Using this approach, an improvement of 22.5% over the maximum ethanol yield previously obtained was achieved, reaching an ethanol yield of 72.4 ± 2.1% of the theoretical maximum by increasing the initial acetate concentration in the fermentation broth.ConclusionsThis study demonstrated high product selectivity towards ethanol using mixed microbial consortia. The thermodynamic analysis carried out proved to be a valuable tool for interpreting the metabolic network of microbial consortia-driven processes and designing microbial-enrichment strategies targeting specific biotransformations.

Effect of ethanol and butanol on autotrophic growth of model homoacetogens.

Research efforts aimed at increasing ethanol and butanol productivity from syngas are currently gaining attention. For most model carboxydotrophic bacteria, production rates, yields and maximum product titres have been studied in detail, but little is known on alcohol toxicity in these bacteria. The aim of this work was to investigate the inhibitory effects of ethanol and butanol on the growth of Clostridium ljungdahlii PETC, C. carboxidivorans P7, and 'Butyribacterium methylotrophicum DSM3468'. Experiments to determine inhibitory effects due to product accumulation were carried out using a synthetic mixture of CO:CO2:H2 as a substrate. These conditions were chosen to mimic gaseous effluents of biomass and waste gasification plants. Inhibition effects were recorded as changes in growth parameters. No significant inhibition was observed for ethanol at concentrations below 15g/L. The three species exhibited higher sensitivity to butanol. Half inhibition constants for butanol could be estimated for P7 (IC50=4.12g/L), DSM3468 (IC50=1.79g/L), and PETC (IC50=9.75g/L). In conclusion, at least for the tested strains, alcohol toxicity is not an immediate handicap for increasing alcohol production of the tested homoacetogenic strains.

A Narrow pH Range Supports Butanol, Hexanol, and Octanol Production from Syngas in a Continuous Co-culture of Clostridium ljungdahlii and Clostridium kluyveri with In-Line Product Extraction.

Carboxydotrophic bacteria (CTB) have received attention due to their ability to synthesize commodity chemicals from producer gas and synthesis gas (syngas). CTB have an important advantage of a high product selectivity compared to chemical catalysts. However, the product spectrum of wild-type CTB is narrow. Our objective was to investigate whether a strategy of combining two wild-type bacterial strains into a single, continuously fed bioprocessing step would be promising to broaden the product spectrum. Here, we have operated a syngas-fermentation process with Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction through gas stripping and product condensing within the syngas recirculation line. The main products from C. ljungdahlii fermentation at a pH of 6.0 were ethanol and acetate at net volumetric production rates of 65.5 and 431 mmol C·L−1·d−1, respectively. An estimated 2/3 of total ethanol produced was utilized by C. kluyveri to chain elongate with the reverse β-oxidation pathway, resulting in n-butyrate and n-caproate at net rates of 129 and 70 mmol C·L−1·d−1, respectively. C. ljungdahlii likely reduced the produced carboxylates to their corresponding alcohols with the reductive power from syngas. This resulted in the longer-chain alcohols n-butanol, n-hexanol, and n-octanol at net volumetric production rates of 39.2, 31.7, and 0.045 mmol C·L−1·d−1, respectively. The continuous production of the longer-chain alcohols occurred only within a narrow pH spectrum of 5.7–6.4 due to the pH discrepancy between the two strains. Regardless whether other wild-type strains could overcome this pH discrepancy, the specificity (mol carbon in product per mol carbon in all other liquid products) for each longer-chain alcohol may never be high in a single bioprocessing step. This, because two bioprocesses compete for intermediates (i.e., carboxylates): (1) chain elongation; and (2) biological reduction. This innate competition resulted in a mixture of n-butanol and n-hexanol with traces of n-octanol.

Butanol tolerance of carboxydotrophic bacteria isolated from manure composts

ABSTRACTCarboxydotrophic bacteria (carboxydotrophs) have the ability to uptake carbon monoxide (CO) and synthesize butanol. The aims of this study were to determine the butanol tolerance and biological production of butanol carboxydotrophic strains. In this study, 11 carboxydotrophic strains were exposed to increasing n-butanol concentrations (1–3% vol/vol) to determine their effect on growth. Butanol production by the strains was quantified and the identity of the strains was elucidated using 16S rRNA sequencing. The carboxydotrophic strains possessed inherent tolerance to butanol and tolerated up to 3% n-butanol. Among the 11 strains, T1-16, M2-32 and M3-28 were the most tolerant to butanol. The 16S rRNA gene sequence of these strains was similar (99% nucleotide similarity) to the butanol-tolerant strains Bacillus licheniformis YP1A, Pediococcus acidilacti IMUA20068 and Enterococcus faecium IMAU60169, respectively. The carboxydotrophic strains screened in this study have two distinct features: (1) high tolerance to butanol and (2) natural production of low concentration of butanol from CO, which distinguish them from other screened butanol-tolerant strains. The butanol tolerance of these carboxydotrophic strains makes them ideal for genetic studies, particularly the molecular mechanisms that enable them to survive such hostile environmental conditions and the identification of genes that confer tolerance to butanol.

Anaerobic carboxydotrophic bacteria in geothermal springs identified using stable isotope probing

Carbon monoxide (CO) is a potential energy and carbon source for thermophilic bacteria in geothermal environments. Geothermal sites ranging in temperature from 45 to 65°C were investigated for the presence and activity of anaerobic CO-oxidizing bacteria. Anaerobic CO oxidation potentials were measured at up to 48.9 μmoles CO g−1 (wet weight) day−1 within five selected sites. Active anaerobic carboxydotrophic bacteria were identified using 13CO DNA stable isotope probing (SIP) combined with pyrosequencing of 16S rRNA genes amplified from labeled DNA. Bacterial communities identified in heavy DNA fractions were predominated by Firmicutes, which comprised up to 95% of all sequences in 13CO incubations. The predominant bacteria that assimilated 13C derived from CO were closely related (>98% 16S rRNA gene sequence identity) to genera of known carboxydotrophs including Thermincola, Desulfotomaculum, Thermolithobacter, and Carboxydocella, although a few species with lower similarity to known bacteria were also found that may represent previously unconfirmed CO-oxidizers. While the distribution was variable, many of the same OTUs were identified across sample sites from different temperature regimes. These results show that bacteria capable of using CO as a carbon source are common in geothermal springs, and that thermophilic carboxydotrophs are probably already quite well known from cultivation studies.

Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter.

How can alcohol production be improved in carboxydotrophic clostridia?

Clostridia are well-known organisms within the scientific community, and many aspects of their life cycle have been studied. Autotrophic strains are especially interesting as microbial cell factories for industrial purposes. Carboxydotrophic clostridia can use CO2/H2 and/or CO as their only carbon and reducing power sources, and they produce alcohols and acids. Both products can be used as either biofuels or feedstock chemicals for many industrial processes, confirming the control of carboxydotrophic clostridia metabolism as one of the most profitable scientific ambitions to be explored. Here, we review the current state of research on alcohol production by carboxydotrophic clostridia, including a comprehensive overview of their metabolic pathways and the key experimental variables that govern alcohol production. Additionally, the genetic and genomic tools for selective and enhanced solvent production, which are currently under intense development, are discussed. In this context, this review covers the main genetic engineering methods that have been utilized to improve the capabilities of solventogenic carboxydotrophic bacteria.

Integrating syngas fermentation with the carboxylate platform and yeast fermentation to reduce medium cost and improve biofuel productivity

To ensure economic implementation of syngas fermentation as a fuel-producing platform, engineers and scientists must both lower operating costs and increase product value. A considerable part of the operating costs is spent to procure chemicals for fermentation medium that can sustain sufficient growth of carboxydotrophic bacteria to convert synthesis gas (syngas: carbon monoxide, hydrogen, and carbon dioxide) into products such as ethanol. Recently, we have observed that wild-type carboxydotrophic bacteria (including Clostridium ljungdahlii) can produce alcohols with a longer carbon chain than ethanol via syngas fermentation when supplied with the corresponding carboxylic acid precursors, resulting in possibilities of increasing product value. Here, we evaluated a proof-of-concept system to couple syngas fermentation with the carboxylate platform to both lower medium costs and increase product value. Our carboxylate platform concept consists of an open culture, anaerobic fermentor that is fed with corn beer from conventional yeast fermentation in the corn kernel-to-ethanol industry. The mixed-culture anaerobic fermentor produces a mixture of carboxylic acids at dilute concentrations within the carboxylate platform effluent (CPE). Besides providing carboxylic acid precursors, this effluent may represent an inexpensive growth medium. An elemental analysis demonstrated that the CPE lacked certain essential trace metals, but contained ammonium, phosphate, sodium, chloride, potassium, magnesium, calcium, and sulphate at required concentrations. CPE medium with the addition of a trace metal solution supported growth and alcohol production of C. ljungdahlii at similar or better levels compared with an optimized synthetic medium (modified ATCC 1754 medium). Other expensive supplements, such as yeast extract or macro minerals (ammonium, phosphate), were not required. Finally, n-butyric acid and n-caproic acid within the CPE were converted into their corresponding medium-chain alcohols n-butanol and n-hexanol.

Quantitative detection of carboxydotrophic bacteria Carboxydothermus in a hot aquatic environment

Quantitative detection of carboxydotrophic bacteria Carboxydothermus in a hot aquatic environment

Biocatalytic reduction of short‐chain carboxylic acids into their corresponding alcohols with syngas fermentation

Short-chain carboxylic acids generated by various mixed- or pure-culture fermentation processes have been considered valuable precursors for production of bioalcohols. While conversion of carboxylic acids into alcohols is routinely performed with catalytic hydrogenation or with strong chemical reducing agents, here, a biological conversion route was explored. The potential of carboxydotrophic bacteria, such as Clostridium ljungdahlii and Clostridium ragsdalei, as biocatalysts for conversion of short-chain carboxylic acids into alcohols, using syngas as a source of electrons and energy is demonstrated. Acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and n-caproic acid were converted into their corresponding alcohols. Furthermore, biomass yields and fermentation stoichiometry from the experimental data were modeled to determine how much metabolic energy C. ljungdahlii generated during syngas fermentation. An ATP yield of 0.4-0.5 mol of ATP per mol CO consumed was calculated in the presence of hydrogen. The ratio of protons pumped across the cell membrane versus electrons transferred from ferredoxin to NAD(+) via the Rnf complex is suggested to be 1.0. Based on these results, we provide suggestions how n-butyric acid to n-butanol conversion via syngas fermentation can be further improved.

Microbiology and genetics of CO utilization in mycobacteria

Although extensive studies on the oxidation of carbon monoxide (CO) in aerobic carboxydotrophic bacteria have been carried out for over 30years, utilization of CO as a source of carbon and energy by mycobacteria was recognized only recently. Studies on pathogenic and nonpathogenic mycobacteria have revealed that the basis for CO utilization in these bacteria is different in many aspects from that of other aerobic carboxydobacteria. We review the basis for CO utilization in mycobacterial carboxydobacteria, which is unique from physiological, biochemical, molecular, genetic and phylogenetic points of view.

Microbial oxidation of methane, ammonium and carbon monoxide, and turnover of nitrous oxide and nitric oxide in soils

The effect of soil microbial processes on production and/or consumption of atmospheric trace gases was studied in four different soils which were preincubated in the presence of elevated concentrations of CH4, NH 4 + or CO, to simulate the growth of the resident populations of methanotrophic, nitrifying, or carboxydotrophic bacteria, respectively. Oxidation of CH4, both at atmospheric (1.8 ppmv) and at elevated (3500 ppmv) CH4 mixing ratios, was stimulated after preincubation with CH4, but not with NH 4 + or CO, indicating that CH4 was oxidized by methanotrophic, but not by nitrifying or carboxydotrophic bacteria. However, the oxidation of CH4 was partially inhibited by addition of NH 4 + and CO. Analogously, oxidation of NH 4 + was partially inhibited by addition of CH4. Oxidation of CO at elevated mixing ratios (2300 ppmv) was stimulated after preincubation with CO, indicating oxidation by carboxydotrophs, but was also stimulated at a small extent after preincubation with CH4, suggesting the involvement of methanotrophs. At atmospheric CO mixing ratios (0.13 ppmv), on the other hand, oxidation of CO was stimulated after preincubation with NH 4 + , indicating that the activity was due to nitrifiers. NO uptake was stimulated in soils preincubated with CH4, indicating the involvement of methanotrophs. However, production of N2O was only stimulated, if CH4 was added as a substrate. The results indicate that especially the methanotrophic and nitrifying populations in soil not only oxidize their specific substrates, but are also involved in the metabolism of other compounds.

Genes encoding ribulosebisphosphate carboxylase and phosphoribulokinase are duplicated in Pseudomonas carboxydovorans and conserved in carboxydotrophic bacteria

Heterologous gene probes derived from cfxLp and cfxPp genes of Alcaligenes eutrophus H16 revealed the presence of structural genes encoding ribulosebisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) on the genome of carboxydotrophic bacteria. The two genes were found to be rather conserved. In Pseudomonas carboxydovorans OM5 cfx genes reside on the plasmid pHCG3 and the chromosome as well, indicating that they are duplicated. Also in all plasmidharboring carboxydotrophic bacteria cfxL and cfxP structural genes were found to be plasmid-coded. Our results extend the list of carboxydotrophy structural genes residing on the plasmid pHCG3 and strongly support the idea that the components essential for the chemolithoautotrophic utilization of CO by Pseudomonas carboxydovorans OM5 are plasmid-coded. A cfxL gene probe from Rhodospirillum rubrum did not detectably hybridize with DNA from any of the carboxydotrophic bacteria examined.

Anaerobic extremely thermophilic carboxydotrophic bacteria in hydrotherms of Kuril Islands

A new group of extremely thermophilic, obligately anaerobic, carboxydotrophic eubacteria is described. The organisms are characterized by a novel type of chemotrophic metabolism in thermophilic environments. They grow at temperatures up to 80-85°C chemolithotrophically with 100% CO in the gas phase as the sole energy source. The CO oxidation is coupled to H2 and CO2 formation according to the equation CO+H2O → H2+CO2. No other products of metabolism are produced. The group of CO-utilizing, H2-producing anaerobes includes diverse bacteria. They are non-sporeforming rods differing in morphology, CO uptake rates, habitats, and maximum growth temperatures. The new carboxydotrophic thermophilic anaerobes are widely distributed in freshwater and coastal marine hydrotherms of the Kuril Islands.

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H 2 -Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H(2)-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 +/- 0.19 mol%), a phospholipid pattern of type II, MK-9(H(4)) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t(d) = 8 h), H(2) plus CO(2) (t(d) = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H(2), nickel was not necessary. The optimum growth temperature was 65 degrees C; no growth was observed below 40 degrees C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70 degrees C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K(m) was 88 mul of CO liter and V(max) was 20.2 mul of CO h mg of protein. The threshold value of S. thermoautotrophicus of 0.2 mul of CO liter was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 mumol min mg of protein. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.