Complete genome sequence of Methanothermobacter sp. DP, a hydrogenotrophic and thermophilic methanogen.

Here, we report the complete genome sequence of the thermophilic hydrogenotrophic methanogen Methanothermobacter sp. DP isolated in South Korea from an anaerobic digester fed with cigarette waste. The genome consists of 1,693,285 bp, with 1,772 protein-coding genes and a GC content of 48.8%.

Chain elongation process for caproate production using lactate as electron donor in Megasphaera hexanoica.

Megasphaera hexnaoica is anaerobic bacteria who has well running reverse ß-oxidation pathway. In previous study, the strain showed excellent production of medium chain carboxylic acids (MCCAs) using fructose as electron donor. In this study, chain elongation process study using lactate instead of fructose was conducted in M. hexnaoica fermentation. It was found that M. hexanoica can use lactate as electron donor in chain elongation process. 8.9 g/L caproate production was achieved in fermentation using lactate as sole electron donor. Compare to fructose condition, lactate as electron donor showed more than 3 times higher specific titer and specific productivity. In addition, when fructose and lactate were used as electron donor simultaneously, further improvement of MCCAs production was observed to achieve maximum caproate productivity of 20.9 g/L/day. Utilization of lactate as electron donor in M. hexanoica showed potential opportunity in chain elongation process.

Complete Genome Sequence of Methanothermobacter sp. Strain THM-1, a Thermophilic and Hydrogenotrophic Methanogen Isolated from an Anaerobic Reactor in South Korea.

Methanothermobacter sp. strain THM-1, a thermophilic and hydrogenotrophic methanogen, was isolated from an anaerobic reactor enriched with thermophilic methanogens. The genome of THM-1 shares 98.81% of its sequence with Methanothermobacter wolfeii isolate SIV6 and consists of 1,724,502 bp with 1,665 protein-coding genes, 50 noncoding RNAs, and a GC content of 48.6%.

Characterisation of bacterial nanocellulose and nanostructured carbon produced from crude glycerol by Komagataeibacter sucrofermentans.

Bacterial nanocellulose (BNC), which has tunable properties, is a precursor of nanostructured energy storage materials; however, the cost of BNC production is challenging. This study uses crude glycerol from the biodiesel industry as a carbon nutrient and first-time carbonised BNC from K. sucrofermentans that is applied in energy storage. From crude glycerol in static cultivation, 6.4 g L-1 BNC was produced with a high crystallinity index (85%) and tensile properties in comparison to conventionally used pure carbon substrates. Carbon materials were derived from the BNC retained fibrous and crystalline features with disordered porous structures. The electrochemical properties of the carbon materials have a specific capacitance of 140 F g-1. This study highlights the valorisation of waste glycerol from the biodiesel industry as a substrate for efficient BNC production and the energy storage potential of carbon derived from BNC as renewable energy materials.

Anaerobic co-digestion of bioplastics as a sustainable mode of waste management with improved energy production - A review.

The world of bioplastics has expanded rapidly in recent decades, and the new waste stream generated is creating major barriers to waste processing. Anaerobic co-digestion is to be considered one of the best options for the efficient processing of bioplastic waste due to its minimal space requirements, lower degrees of environmental pollution, and renewable energy generation. The higher carbon to nitrogen (C/N) ratio of bioplastics poses a challenge to anaerobic digestion, but co-digestion with lower C/N ratio biowastes can efficiently degrade bioplastics and improve biogas production in the system. In the future, the collection of organic waste in biodegradable plastic bags makes the waste management process easier for anaerobic digestion plants. The present review paper discusses current trends of bioplastic usage, degradation strategies, and the potential of anaerobic co-digestion for waste management with improved energy production in anaerobic digesters.

Impact of feedstocks and downstream processing technologies on the economics of caproic acid production in fermentation by Megasphaera elsdenii T81.

Caproic acid (CA) was produced by Megasphaera elsdenii T81 with Jerusalem artichoke tubers (JA) as a feedstock. More CA was produced under the medium with the acid hydrolysate of JA than the comparative medium with a carbon composition similar to that of JA. CA was produced up to 13.0 g/L and 0.52 g/L/h with extractive fermentation using a mixed solvent of alamine 336 in oleyl alcohol at 37 °C. The JA cost to produce 1 ton of CA is only 505 USD, which is much lower than that required for purchasing sucrose (860 USD) in CA production. As a result of the analysis performed using SuperPro Designer, including the cost of distillation to obtain pure CA, the estimated production cost for CA from dry JA is 1869 USD/ton CA at the production scale of 2000 ton/year, which is lower than the current market price for petroleum-derived CA (~2500 USD/ton).

Pretreatment strategies for enhanced biogas production from lignocellulosic biomass.

The inclusion of a pretreatment step in anaerobic digestion processes increases the digestibility of lignocellulosic biomass and enhances biogas yields by promoting lignin removal and the destruction of complex biomass structures. The increase in surface area enables the efficient interaction of microbes or enzymes, and a reduction in cellulose crystallinity improves the digestion process under anaerobic conditions. The pretreatment methods may vary based on the type of the lignocellulosic biomass, the nature of the subsequent process and the overall economics of the process. An improved biogas production by 1200% had been reported when ionic liquid used as pretreatment strategy for anaerobic digestion. The different pretreatment techniques used for lignocellulosic biomasses are generally grouped into physical, chemical, physicochemical, and biological methods. These four modes of pretreatment on lignocellulosic biomass and their impact on biogas production process is the major focus of this review article.

Complete Genomic Sequence of the Thermophilic and Hydrogenotrophic Methanogen Methanothermobacter sp. Strain KEPCO-1.

Here, we describe the complete genome of Methanothermobacter sp. strain KEPCO-1, a thermophilic and hydrogenotrophic methanogen that was isolated from an anaerobic digester in Seoul, Republic of Korea. The genome of KEPCO-1 shares 96.98% of its sequence with Methanothermobacter marburgensis strain DSM 2133 and consists of 1,741,029 bp, with 1,822 protein-coding genes, 44 noncoding RNAs, and a GC content of 48.47%. The development of this genome will facilitate future genomic studies of KEPCO-1.

Simultaneous production of methane and acetate by thermophilic mixed culture from carbon dioxide in bioelectrochemical system.

A thermophilic bioelectrochemical system was operated with mixed culture at 60 °C, while introducing only carbon dioxide. Methane production was initially observed in a membrane-less single chamber without a mediator, but eventually acetate was also found as 10.5 g/L after 137 days of operation. Comparing the microbial communities before and after the electricity supply using next-generation sequencing technology, acetoclastic methanogens such as Methanosaeta concilii were increased, and this result also indicates the production of acetate in bioelectrochemical CO2 conversion system. With the advent of sulfate-reducing bacteria, Desulfotomaculum peckii was considered to be an acetate production promoter. These high production results for both methane and acetate can be applied to CO2 storage using excess electricity for value-added chemicals.

Enhanced extraction of butyric acid under high-pressure CO2 conditions to integrate chemical catalysis for value-added chemicals and biofuels.

BACKGROUND: Extractive fermentation with the removal of carboxylic acid requires low pH conditions because acids are better partitioned into the solvent phase at low pH values. However, this requirement conflicts with the optimal near-neutral pH conditions for microbial growth. RESULTS: CO2 pressurization was used, instead of the addition of chemicals, to decrease pH for the extraction of butyric acid, a fermentation product of Clostridium tyrobutyricum, and butyl butyrate was selected as an extractant. CO2 pressurization (50 bar) improved the extraction efficiency of butyric acid from a solution at pH 6, yielding a distribution coefficient (D) 0.42. In situ removal of butyric acid during fermentation increased the production of butyric acid by up to 4.10 g/L h, an almost twofold increase over control without the use of an extraction process. CONCLUSION: In situ extraction of butyric acid using temporal CO2 pressurization may be applied to an integrated downstream catalytic process for upgrading butyric acid to value-added chemicals in an organic solvent.

Pyrosequencing analysis of microbial communities in hollow fiber-membrane biofilm reactors system for treating high-strength nitrogen wastewater.

Wastewaters from swine farms, nitrogen-dealing industries or side-stream processes of a wastewater treatment plant (e.g., anaerobic digesters, sludge thickening processes, etc.) are characterized by low C/N ratios and not easily treatable. In this study, a hollow fiber-membrane biofilm reactors (HF-MBfR) system consisting of an O2-based HF-MBfR and an H2-based HF-MBfR was applied for treating high-strength wastewater. The reactors were continuously operated with low supply of O2 and H2 and without any supply of organic carbon for 250 d. Gradual increase of ammonium and nitrate concentration in the influent showed stable and high nitrogen removal efficiency, and the maximum ammonium and nitrate removal rates were 0.48 kg NH4(+)-N m(-3) d(-1) and 0.55 kg NO3(-)-N m(-3) d(-1), respectively. The analysis of the microbial communities using pyrosequencing analysis indicated that Nitrosospira multiformis, ammonium-oxidizing bacteria, and Nitrobacter winogradskyi and Nitrobacter vulgaris, nitrite-oxidizing bacteria were highly enriched in the O2-based HF-MBfR. In the H2-based HF-MBfR, hydrogenotrophic denitrifying bacteria belonging to the family of Thiobacillus and Comamonadaceae were initially dominant, but were replaced to heterotrophic denitrifiers belonging to Rhodocyclaceae and Rhodobacteraceae utilizing by-products induced from autotrophic denitrifying bacteria. The pyrosequencing analysis of microbial communities indicates that the autotrophic HF-MBfRs system well developed autotrophic nitrifying and denitrifying bacteria within a relatively short period to accomplish almost complete nitrogen removal.

Production of medium-chain carboxylic acids by Megasphaera sp. MH with supplemental electron acceptors.

BACKGROUND: C5-C8 medium-chain carboxylic acids are valuable chemicals as the precursors of various chemicals and transport fuels. However, only a few strict anaerobes have been discovered to produce them and their production is limited to low concentrations because of product toxicity. Therefore, a bacterial strain capable of producing high-titer C5-C8 carboxylic acids was strategically isolated and characterized for production of medium chain length carboxylic acids. RESULTS: Hexanoic acid-producing anaerobes were isolated from the inner surface of a cattle rumen sample. One of the isolates, displaying the highest hexanoic acid production, was identified as Megasphaera sp. MH according to 16S rRNA gene sequence analysis. Megasphaera sp. MH metabolizes fructose and produces various medium-chain carboxylic acids, including hexanoic acid, in low concentrations. The addition of acetate to the fructose medium as an electron acceptor increased hexanoic acid production as well as cell growth. Supplementation of propionate and butyrate into the medium also enhanced the production of C5-C8 medium-chain carboxylic acids. Megasphaera sp. MH produced 5.7 g L(-1) of pentanoic acid (C5), 9.7 g L(-1) of hexanoic acid (C6), 3.2 g L(-1) of heptanoic acid (C7) and 1.2 g L(-1) of octanoic acid (C8) in medium supplemented with C2-C6 carboxylic acids as the electron acceptors. This is the first report on the production of high-titer heptanoic acid and octanoic acid using a pure anaerobic culture. CONCLUSION: Megasphaera sp. MH metabolized fructose for the production of C2-C8 carbon-chain carboxylic acids using various electron acceptors and achieved a high-titer of 9.7 g L(-1) and fast productivity of 0.41 g L(-1) h(-1) for hexanoic acid. However, further metabolic activities of Megaspahera sp. MH for C5-C8 carboxylic acids production must be deciphered and improved for industrially relevant production levels.

Extracellular electron transfer from cathode to microbes: application for biofuel production.

Extracellular electron transfer in microorganisms has been applied for bioelectrochemical synthesis utilizing microbes to catalyze anodic and/or cathodic biochemical reactions. Anodic reactions (electron transfer from microbe to anode) are used for current production and cathodic reactions (electron transfer from cathode to microbe) have recently been applied for current consumption for valuable biochemical production. The extensively studied exoelectrogenic bacteria Shewanella and Geobacter showed that both directions for electron transfer would be possible. It was proposed that gram-positive bacteria, in the absence of cytochrome C, would accept electrons using a cascade of membrane-bound complexes such as membrane-bound Fe-S proteins, oxidoreductase, and periplasmic enzymes. Modification of the cathode with the addition of positive charged species such as chitosan or with an increase of the interfacial area using a porous three-dimensional scaffold electrode led to increased current consumption. The extracellular electron transfer from the cathode to the microbe could catalyze various bioelectrochemical reductions. Electrofermentation used electrons from the cathode as reducing power to produce more reduced compounds such as alcohols than acids, shifting the metabolic pathway. Electrofuel could be generated through artificial photosynthesis using electrical energy instead of solar energy in the process of carbon fixation.

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane.

Hydrogenotrophic methanogens can use gaseous substrates, such as H2 and CO2, in CH4 production. H2 gas is used to reduce CO2. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH4 production from CO2 and H2. CO2 and H2 were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5-5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH4 production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH4 production process. The results show that acidic operation of a CH4 production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.

Analysis of Microbial Communities in Biofilms from CSTR-Type Hollow Fiber Membrane Biofilm Reactors for Autotrophic Nitrification and Hydrogenotrophic Denitrification.

Two hollow fiber membrane biofilm reactors (HF-MBfRs) were operated for autotrophic nitrification and hydrogenotrophic denitrification for over 300 days. Oxygen and hydrogen were supplied through the hollow fiber membrane for nitrification and denitrification, respectively. During the period, the nitrogen was removed with the efficiency of 82-97% for ammonium and 87-97% for nitrate and with the nitrogen removal load of 0.09-0.26 kg NH4(+)-N/m(3)/d and 0.10-0.21 kg NO3(-)-N/m(3)/d, depending on hydraulic retention time variation by the two HF-MBfRs for autotrophic nitrification and hydrogenotrophic denitrification, respectively. Biofilms were collected from diverse topological positions in the reactors, each at different nitrogen loading rates, and the microbial communities were analyzed with partial 16S rRNA gene sequences in denaturing gradient gel electrophoresis (DGGE). Detected DGGE band sequences in the reactors were correlated with nitrification or denitrification. The profile of the DGGE bands depended on the NH4(+) or NO3(-) loading rate, but it was hard to find a major strain affecting the nitrogen removal efficiency. Nitrospira-related phylum was detected in all biofilm samples from the nitrification reactors. Paracoccus sp. and Aquaspirillum sp., which are an autohydrogenotrophic bacterium and an oligotrophic denitrifier, respectively, were observed in the denitrification reactors. The distribution of microbial communities was relatively stable at different nitrogen loading rates, and DGGE analysis based on 16S rRNA (341f /534r) could successfully detect nitrate-oxidizing and hydrogen-oxidizing bacteria but not ammonium-oxidizing bacteria in the HF-MBfRs.

Butyric acid production from red algae by a newly isolated Clostridium sp. S1.

OBJECTIVE: To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required. RESULTS: A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5-2 g levulinic acid/l and recovered from HMF inhibition at 0.6-2.5 g/l, resulting in 85-92% butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l. CONCLUSION: Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

Electrochemical detoxification of phenolic compounds in lignocellulosic hydrolysate for Clostridium fermentation.

Lignocellulosic biomass is being preferred as a feedstock in the biorefinery, but lignocellulosic hydrolysate usually contains inhibitors against microbial fermentation. Among these inhibitors, phenolics are highly toxic to butyric acid-producing and butanol-producing Clostridium even at a low concentration. Herein, we developed an electrochemical polymerization method to detoxify phenolic compounds in lignocellulosic hydrolysate for efficient Clostridium fermentation. After the electrochemical detoxification for 10h, 78%, 77%, 82%, and 94% of p-coumaric acid, ferulic acid, vanillin, and syringaldehyde were removed, respectively. Furthermore, 71% of total phenolics in rice straw hydrolysate were removed without any sugar-loss. Whereas the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were completely inhibited in un-detoxified hydrolysate, those in detoxifying rice straw hydrolysate were recovered to 70-100% of the control cultures. The electrochemical detoxification method described herein provides an efficient strategy for producing butanol and butyric acid through Clostridium fermentation with lignocellulosic hydrolysate.

Electricity-driven metabolic shift through direct electron uptake by electroactive heterotroph Clostridium pasteurianum.

Although microbes directly accepting electrons from a cathode have been applied for CO2 reduction to produce multicarbon-compounds, a high electron demand and low product concentration are critical limitations. Alternatively, the utilization of electrons as a co-reducing power during fermentation has been attempted, but there must be exogenous mediators due to the lack of an electroactive heterotroph. Here, we show that Clostridium pasteurianum DSM 525 simultaneously utilizes both cathode and substrate as electron donors through direct electron transfer. In a cathode compartment poised at +0.045 V vs. SHE, a metabolic shift in C. pasteurianum occurs toward NADH-consuming metabolite production such as butanol from glucose (20% shift in terms of NADH consumption) and 1,3-propandiol from glycerol (21% shift in terms of NADH consumption). Notably, a small amount of electron uptake significantly induces NADH-consuming pathways over the stoichiometric contribution of the electrons as reducing equivalents. Our results demonstrate a previously unknown electroactivity and metabolic shift in the biochemical-producing heterotroph, opening up the possibility of efficient and enhanced production of electron-dense metabolites using electricity.

Butyrate production enhancement by Clostridium tyrobutyricum using electron mediators and a cathodic electron donor.

Electron mediators and electron supply through a cathode were examined to enhance the reducing power for butyrate production by an acidogenic clostridium strain, Clostridium tyrobutyricum BAS 7. Among the tested electron mediators, methyl viologen (MV)-amended cultures showed an increase of butyrate productivity (1.3 times), final concentration (1.4 times), and yield (1.3 times). The electron flow altered by MV addition from the ferredoxin pool to the NADH pool was shown by one electron model, implying that more available NADH increased butyrate production. In the cathode compartment poised at -400 mV versus the Ag/AgCl electrode, the neutral red (NR)-amended cultures of Clostridium tyrobutyricum BAS 7 increased butyrate concentration (from 5 to 8.8 g/L) and yield (from 0.33 up to 0.44 g/g) with no acetate production at all. Given that electrically reduced NR (NR(red) , yellow) by the cathode was re-oxidized (NR(ox) , red) in the cells on the basis of color change, electron flow from NR(red) to NAD(+) (i.e., NADH generation) induced an increase in butyrate production. This is the first report to show the increase of butyric acid production by electrically driven acidogenesis. These results show that the electron flow altered NADH formation by electron mediators and by the cathodic electron donor, increasing the yield and selectivity of reduced end-products like butyrate.

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme.

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 µA cm(-1) by adding 1mM glucose at an applied potential of -0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67mM (S/N=3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.