Variability in n-caprylate and n-caproate producing microbiomes in reactors with in-line product extraction.

Medium-chain carboxylates (MCCs) are used in various industrial applications. These chemicals are typically extracted from palm oil, which is deemed not sustainable. Recent research has focused on microbial chain elongation using reactors to produce MCCs, such as n-caproate (C6) and n-caprylate (C8), from organic substrates such as wastes. Even though the production of n-caproate is relatively well-characterized, bacteria and metabolic pathways that are responsible for n-caprylate production are not. Here, three 5 L reactors with continuous membrane-based liquid-liquid extraction (i.e., pertraction) were fed ethanol and acetate and operated for an operating period of 234 days with different operating conditions. Metagenomic and metaproteomic analyses were employed. n-Caprylate production rates and reactor microbiomes differed between reactors even when operated similarly due to differences in H2 and O2 between the reactors. The complete reverse ß-oxidation (RBOX) pathway was present and expressed by several bacterial species in the Clostridia class. Several Oscillibacter spp., including Oscillibacter valericigenes, were positively correlated with n-caprylate production rates, while Clostridium kluyveri was positively correlated with n-caproate production. Pseudoclavibacter caeni, which is a strictly aerobic bacterium, was abundant across all the operating periods, regardless of n-caprylate production rates. This study provides insight into microbiota that are associated with n-caprylate production in open-culture reactors and provides ideas for further work.IMPORTANCEMicrobial chain elongation pathways in open-culture biotechnology systems can be utilized to convert organic waste and industrial side streams into valuable industrial chemicals. Here, we investigated the microbiota and metabolic pathways that produce medium-chain carboxylates (MCCs), including n-caproate (C6) and n-caprylate (C8), in reactors with in-line product extraction. Although the reactors in this study were operated similarly, different microbial communities dominated and were responsible for chain elongation. We found that different microbiota were responsible for n-caproate or n-caprylate production, and this can inform engineers on how to operate the systems better. We also observed which changes in operating conditions steered the production toward and away from n-caprylate, but more work is necessary to ascertain a mechanistic understanding that could be predictive. This study provides pertinent research questions for future work.

The whole-genome sequence of Bacillus sp. KICET-3, isolated from doenjang.

Bacillus sp. KICET-3, isolated from doenjang, a traditional Korean fermented food, has a single chromosomal DNA fragment of 4,616,861 bp, and the G+C content is 45.52%. It is estimated to have 4,450 predicted coding DNA sequences, 84 tRNAs, and 24 rRNAs.

Draft genome sequence of Magnusiomyces sp. LA-1 isolated from a C6-C8 acid-producing bioreactor.

Here, we report the draft genome of Magnusiomyces sp. LA-1, which was isolated from a C6-C8 carboxylic acid-producing bioreactor. The draft genome of Magnusiomyces sp. LA-1 is 19,829,165 bp in length, is divided into six contigs that comprise 6,557 CDS regions, and has a GC content of 34.5%.

Complete Genome Sequence of Bacillus sp. Strain KICET-1, Isolated from Soybean Paste (Doenjang).

Bacillus sp. strain KICET-1, a bacterium isolated from traditional Korean soybean paste (Doenjang) at Osong, has one 4,099,652-bp DNA chromosome. The G+C content is 46.1%, and KICET-1 shares 99.64% similarity with Bacillus velezensis CR-502T (AY603658), according to phylogenetic classification based on 16S rRNA gene sequences.

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%.

Highly Stable and Fine-Textured Hybrid Microspheres for Entrapment of Cosmetic Active Ingredients.

This study details the preparation and application of supramolecular host-guest inclusion complexes entrapping biomineralized microspheres for long-term storage and their pH-responsive behavior. The microspheres were assembled using a CaCO3 synthesis process coupled with cyclodextrin-tetrahydrocurcumin (CD-THC) inclusion complexes, forming fine-textured and mechanically stable hybrid materials. The products were successfully characterized using field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and particle size analysis (PSA). Various parameters such as the Brunauer-Emmett-Teller (BET) surface area, single point total pore volume, and pore size via adsorption/desorption analysis were also determined. The obtained THC-entrapped hybrid microspheres contained as high as 20 wt % THC loading and were very stable, preserving 90% of the initial concentration over four weeks of storage at different temperatures, largely limiting THC leaching and indicating high stability in a physiological environment. In addition, the pH-responsive release of THC from the hybrid microspheres was observed, showing potential use for application to weakly acidic skin surfaces. To our knowledge, this is the first demonstration of antiaging cosmetic formulation technology using biomineralization based on the co-synthesis of CaCO3 and CD-THC complexes.

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).

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.

The Isolate Caproiciproducens sp. 7D4C2 Produces n-Caproate at Mildly Acidic Conditions From Hexoses: Genome and rBOX Comparison With Related Strains and Chain-Elongating Bacteria.

Bulk production of medium-chain carboxylates (MCCs) with 6-12 carbon atoms is of great interest to biotechnology. Open cultures (e.g., reactor microbiomes) have been utilized to generate MCCs in bioreactors. When in-line MCC extraction and prevention of product inhibition is required, the bioreactors have been operated at mildly acidic pH (5.0-5.5). However, model chain-elongating bacteria grow optimally at neutral pH values. Here, we isolated a chain-elongating bacterium (strain 7D4C2) that grows at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse ß-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n-butyrate, n-caproate, biomass, and H2/CO2 from hexoses. With only fructose as substrate (pH 5.5), the maximum n-caproate specificity (i.e., products per other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and n-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and n-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing n-caproate specificity from ∼92% at mildly acidic pH (pH 4.5-5.0) to ∼24% at alkaline pH (pH 9.0). Moreover, when carboxylates were extracted from the broth (undissociated n-caproic acid was ∼0.3 mM), the n-caproate selectivity (i.e., product per substrate fed) was 42.6 ± 19.0% higher compared to 7D4C2 cultures without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolates Caproicibacter fermentans (99.5%) and Caproiciproducens galactitolivorans (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, C. fermentans, and C. galactitolivorans belong to the same genus of Caproiciproducens. Their rBOX genes are conserved and located next to each other, forming a gene cluster, which is different than for other chain-elongating bacteria such as Megasphaera spp. In conclusion, Caproiciproducens spp., comprising strain 7D4C2, C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. Caproiciproducens sp. 7D4C2 (DSM 110548) was studied here to understand n-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into n-caproate.

An Efficient New Process for the Selective Production of Odd-Chain Carboxylic Acids by Simple Carbon Elongation Using Megasphaera hexanoica.

The caproate-producing bacterium, Megasphaera hexanoica, metabolizes fructose to produce C2~C8 carbon-chain carboxylic acids using various electron acceptors. In particular, odd-chain carboxylic acids (OCCAs) such as valerate (C5) and heptanoate (C7), were produced at relatively high concentrations upon propionate supplementation. Using a statistical experimental design method, the optimal culture medium was established for the selective production of OCCAs among the total produced acids. In a medium containing 2.42 g L-1 sodium acetate and 18.91 g L-1 sodium propionate, M. hexanoica produced 9.48 g L-1 valerate, 2.48 g L-1 heptanoate, and 0.12 g L-1 caproate. To clarify the metabolism of the exogenous added propionate for OCCAs production, 13C tracer experiments were performed by supplementing the culture broth with [1,2,3-13C3] propionate. The metabolites analysis based on mass spectrometry showed that the propionate was only used to produce valerate and heptanoate without being participated in other metabolic pathways. Furthermore, the carbon elongation pathway in M. hexanoica was explained by the finding that the incorporation of propionate and acetate in the produced valerate occurred in only one orientation.

New coculture system of Clostridium spp. and Megasphaera hexanoica using submerged hollow-fiber membrane bioreactors for caproic acid production.

In this study, a coculture bioprocess was developed with Clostridium strains producing butyric acid and Megasphaera hexanoica producing caproic acid from the butyric acid. The two bacterial strains were each cultivated in two submerged hollow-fiber membrane bioreactors (s-HF/MBRs), separately. Each fermentation broth was filtered through the membrane modules, and the filtered broth was either interchanged on another reactor or obtained sequentially through. Using s-HF/MBRs, the caproic acid concentration increased to 10.08 g L-1, with the fastest productivity of 0.69 g L-1 h-1, which higher than that previously reported.

Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing.

The aim of this study is to demonstrate that rebalancing of metabolic fluxes at acetyl-CoA branch node can substantially improve the titer and productivity of hexanoic acid in recombinant Escherichia coli strains. First, a hexanoic acid-producing E. coli strain was constructed by expressing genes encoding ß-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Next, metabolic flux was optimized at the acetyl-CoA branch node by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Four synthetic 5'-untranslated regions were designed for atoB using UTR Designer to modulate the expression level of the gene. Notably, the productivity of the optimized strain (14.7 mg/L/h) was the highest among recombinant E. coli strains in literature when using a similar inoculum size for fermentation. These results show that fine-tuning the expression level of atoB is critical for production of hexanoic acid.

Megasphaera hexanoica sp. nov., a medium-chain carboxylic acid-producing bacterium isolated from a cow rumen.

Strain MHT, a strictly anaerobic, Gram-stain-negative, non-spore-forming, spherical coccus or coccoid-shaped microorganism, was isolated from a cow rumen during a screen for hexanoic acid-producing bacteria. The microorganism grew at 30-40 °C and pH 5.5-7.5 and exhibited production of various short- and medium-chain carboxylic acids (acetic acid, butyric acid, pentanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid and octanoic acid), as well as H2 and CO2 as biogas. Phylogenetic analysis based on 16S rRNA gene sequencing demonstrated that MHT represents a member of the genus Megasphaera, with the closest relatives being Megapsphaera indica NMBHI-10T (94.1 % 16S rRNA sequence similarity), Megasphaera elsdenii DSM 20460T (93.8 %) and Megasphaera paucivorans DSM 16981T (93.8 %). The major cellular fatty acids produced by MHT included C12 : 0, C16 : 0, C18 : 1cis 9, and C18 : 0, and the DNA G+C content of the MHT genome is 51.8 mol%. Together, the distinctive phenotypic and phylogenetic characteristics of MHT indicate that this microorganism represents a novel species of the genus Megasphaera, for which the name Megasphaera hexanoica sp. nov. is herein proposed. The type strain of this species is MHT (=KCCM 43214T=JCM 31403T).

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.

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.

In situ extractive fermentation for the production of hexanoic acid from galactitol by Clostridium sp. BS-1.

Clostridium sp. BS-1 produces hexanoic acid as a metabolite using galactitol and enhanced hexanoic acid production was obtained by in situ extractive fermentation with Clostridium sp. BS-1 under an optimized medium composition. For medium optimization, five ingredients were selected as variables, and among them yeast extract, tryptone, and sodium butyrate were selected as significant variables according to a fractional factorial experimental design, a steepest ascent experimental design, and a Box-Behnken experimental design. The optimized medium had the following compositions in modified Clostridium acetobutyricum (mCAB) medium: 15.5gL(-1) of yeast extract, 10.13gL(-1) of tryptone, 0.04gL(-1) of FeSO4·7H2O, 0.85gL(-1) of sodium acetate, and 6.47gL(-1) of sodium butyrate. The predicted concentration of hexanoic acid with the optimized medium was 6.98gL(-1), and this was validated experimentally by producing 6.96gL(-1) of hexanoic acid with Clostridium sp. BS-1 under the optimized conditions. In situ extractive fermentation for hexanoic acid removal was then applied in a batch culture system with the optimized medium and 10% (v/v) alamine 336 in oleyl alcohol as an extractive solvent. The pH of the culture in the extractive fermentation was maintained at 5.4-5.6 by an acid balance between production and retrieval by extraction. During a 16 day culture, the hexanoic acid concentration in the solvent increased to 32gL(-1) while it was maintained in a range of 1-2gL(-1) in the medium. The maximum rate of hexanoic acid production was 0.34gL(-1)h(-1) in in situ extractive fermentation.

In situ biphasic extractive fermentation for hexanoic acid production from sucrose by Megasphaera elsdenii NCIMB 702410.

Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid-liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l⁻¹ hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l⁻¹ when the medium was supplemented by 5 g l⁻¹ sodium butyrate. A biphasic liquid-liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l⁻¹ of hexanoic acid from 54.85 g l⁻¹ of sucrose during 144 h of culture, and 26.52 and 1.90 g l⁻¹ of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l⁻¹ h⁻¹ and 0.50 g g⁻¹ sucrose, respectively.

Co-culturing a novel Bacillus strain with Clostridium tyrobutyricum ATCC 25755 to produce butyric acid from sucrose.

BACKGROUND: Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755T; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755T to ferment sucrose to butyric acid. RESULTS: B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755T to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755T cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h. CONCLUSIONS: Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated.