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We recently proposed a two-stage Power-to-Protein technology to produce microbial protein from renewable electric power and CO 2 . Two stages were operated in series: Clostridium ljungdahlii in Stage A to reduce CO 2 with H 2 into acetate, and Saccharomyces cerevisiae in Stage B to utilize O 2 and produce microbial protein from acetate. Renewable energy can be used to power water electrolysis to produce H 2 and O 2 . A drawback of Stage A was the need for continuous vitamin supplementation. In this study, by using the more robust thermophilic acetogen Thermoanaerobacter kivui instead of C. ljungdahlii, vitamin supplementation was no longer needed. Additionally, S. cerevisiae produced folate when grown with acetate as a sole carbon source, achieving a total folate concentration of 6.7 mg per 100 g biomass with an average biomass concentration of 3 g l -1 . The developed Power-to-Vitamin system enables folate production from renewable power and CO 2 with zero or negative net-carbon emissions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 H 2 and O 2 between the reactors. The complete reverse β-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., Competing Interests: L.T.A. has ownership in Capro-X, Inc., which is a start-up company that is commercializing a chain-elongating biotechnology production platform.

Thermophilic acetogenic bacteria have attracted attention as promising candidates for biotechnological applications such as syngas fermentation, microbial electrosynthesis, and methanol conversion. Here, we aimed to isolate and characterize novel thermophilic acetogens from diverse environments. Enrichment of heterotrophic and autotrophic acetogens was monitored by 16S rRNA gene-based bacterial community analysis. Seven novel Moorella strains were isolated and characterized by genomic and physiological analyses. Two Moorella humiferrea isolates showed considerable differences during autotrophic growth. The M. humiferrea LNE isolate (DSM 117358) fermented carbon monoxide (CO) to acetate, while the M. humiferrea OCP isolate (DSM 117359) transformed CO to hydrogen and carbon dioxide (H2 + CO2), employing the water-gas shift reaction. Another carboxydotrophic hydrogenogenic Moorella strain was isolated from the covering soil of an active charcoal burning pile and proposed as the type strain (ACPsT) of the novel species Moorella carbonis (DSM 116161T and CCOS 2103T). The remaining four novel strains were affiliated with Moorella thermoacetica and showed, together with the type strain DSM 2955T, the production of small amounts of ethanol from H2 + CO2 in addition to acetate. The physiological analyses of the novel Moorella strains revealed isolate-specific differences that considerably increase the knowledge base on thermophilic acetogens for future applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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%., Competing Interests: The authors declare no conflict of interest.

Integrating hydrothermal treatment processes and anaerobic digestion (AD) is promising for maximizing resource recovery from biomass and organic waste. The process water generated during hydrothermal treatment contains high concentrations of organic matter, which can be converted into biogas using AD. However, process water also contains various compounds that inhibit the AD process. Fingerprinting these inhibitors and identifying suitable mitigation strategies and detoxification methods is necessary to optimize the integration of these two technologies. By examining the existing literature, we were able to: (1) compare the methane yields and organics removal efficiency during AD of various hydrothermal treatment process water; (2) catalog the main AD inhibitors found in hydrothermal treatment process water; (3) identify recalcitrant components limiting AD performance; and (4) evaluate approaches to detoxify specific inhibitors and degrade recalcitrant components. Common inhibitors in process water are organic acids (at high concentrations), total ammonia nitrogen (TAN), oxygenated organics, and N-heterocyclic compounds. Feedstock composition is the primary determinant of organic acid and TAN formation (carbohydrates-rich and protein-rich feedstocks, respectively). In contrast, processing conditions (e.g., temperature, pressure, reaction duration) influence the formation extent of oxygenated organics and N-heterocyclic compounds. Struvite precipitation and zeolite adsorption are the most widely used approaches to eliminate TAN inhibition. In contrast, powdered and granular activated carbon and ozonation are the preferred methods to remove toxic substances before AD treatment. Currently, ozonation is the most effective approach to reduce the toxicity and recalcitrance of N and O-heterocyclic compounds during AD. Microaeration methods, which disrupt the AD microbiome less than ozone, might be more practical for nitrifying TAN and degrading recalcitrant compounds, but further research in this area is necessary., (© 2024. The Author(s).)

The ubiquitous use of volatile siloxanes in a myriad of product formulations has led to a widespread distribution of these persistent contaminants in both natural ecosystems and wastewater treatment plants. Microbial degradation under microaerobic conditions is a promising approach to mitigate D4 and D5 siloxanes while recovering energy in wastewater treatment plants. This study examined D4/D5 siloxanes biodegradation under both anaerobic and microaerobic conditions ( [Formula: see text]  = 0, 1, 3 %) using wastewater sludge. Results show that the use of microaeration in an otherwise strictly anaerobic environment significantly enhances siloxane conversion to methane. 16S rRNA gene sequencing identified potential degraders, including Clostridium lituseburense, Clostridium bifermentans and Synergistales species. Furthermore, chemical analysis suggested a stepwise siloxane conversion preceding methanogenesis under microaerobic conditions. This study demonstrates the feasibility of microaerobic siloxane biodegradation, laying groundwork for scalable removal technologies in wastewater treatment plants, ultimately highlighting the importance of using bio-based approaches in tackling persistent pollutants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lactate is among the top-ten-biobased products. It occurs naturally as D- or L-isomer and as a racemic mixture (DL-lactate). Generally, lactate with a high optical purity is more valuable. In searching for suitable renewable feedstocks for lactate production, unutilized organic waste streams are increasingly coming into focus. Here, we investigated acid whey, which is a lactose-rich byproduct of yogurt production, that represents a considerable environmental footprint for the dairy industry. We investigated the steering of the lactate-isomer composition in a continuous and open culture system (HRT = 0.6 d) at different pH values (pH 5.0 vs. pH 6.5) and process temperatures (38°C to 50°C). The process startup was achieved by autoinoculation. At a pH of 5.0 and a temperature of 47°C-50°C, exclusive D-lactate production occurred because of the dominance of Lactobacillus spp. (> 95% of relative abundance). The highest volumetric D-lactate production rate of 722 ± 94.6 mmol C L -1 d -1 (0.90 ± 0.12 g L -1  h -1 ), yielding 0.93 ± 0.15 mmol C mmol C -1 , was achieved at a pH of 5.0 and a temperature of 44°C (n = 18). At a pH of 6.5 and a temperature of 44°C, we found a mixture of DL-lactate (average D-to-L-lactate production rate ratio of 1.69 ± 0.90), which correlated with a high abundance of Streptococcus spp. and Enterococcus spp. However, exclusive L-lactate production could not be achieved. Our results show that for the continuous conversion of lactose-rich dairy waste streams, the pH was a critical process parameter to control the yield of lactate isomers by influencing the composition of the microbiota. In contrast, temperature adjustments allowed the improvement of bioprocess kinetics., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Largus T. Angenent reports financial support was provided by Federal Ministry of Education and Research Berlin Office. Largus T Angenent reports financial support was provided by Alexander von Humboldt Foundation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Methanogenesis allows methanogenic archaea to generate cellular energy for their growth while producing methane. Thermophilic hydrogenotrophic species of the genus Methanothermobacter have been recognized as robust biocatalysts for a circular carbon economy and are already applied in power-to-gas technology with biomethanation, which is a platform to store renewable energy and utilize captured carbon dioxide. Here, we generated curated genome-scale metabolic reconstructions for three Methanothermobacter strains and investigated differences in the growth performance of these same strains in chemostat bioreactor experiments with hydrogen and carbon dioxide or formate as substrates. Using an integrated systems biology approach, we identified differences in formate anabolism between the strains and revealed that formate anabolism influences the diversion of carbon between biomass and methane. This finding, together with the omics datasets and the metabolic models we generated, can be implemented for biotechnological applications of Methanothermobacter in power-to-gas technology, and as a perspective, for value-added chemical production., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH., Competing Interests: The authors declare no conflict of interest.

Recycling waste gases from industry is vital for the transition toward a circular economy. The model microbe Clostridium ljungdahlii reduces carbon from syngas and primarily produces acetate and ethanol. Here, a gas fermentation experiment is presented in chemostats with C. ljungdahlii and pure carbon monoxide (CO) as feedstock while entirely omitting yeast extract. A maximum ethanol production rate of 0.07 ± 0.01 g L -1 h -1 and a maximum average ethanol/acetate ratio of 1.41 ± 0.39 was observed under steady-state conditions. This confirmed that CO as the sole feedstock pushes the metabolism toward more reduced fermentation products. This effect was even more pronounced when 15 mM sodium acetate was added to the feed medium. An ethanol production rate of 0.23 ± 0.01 g L -1 h -1 was achieved, representing an increase of more than 240%. This increase was accompanied by an increase in cell density and selectivity toward ethanol, with a maximum average ethanol/acetate ratio of 92.96 ± 28.39. Oxygen contaminations voided this effect, although the cultures were still able to maintain a stable biomass concentration and ethanol production rate. These findings highlight the potential of CO-fermentation with acetate augmentation and the importance of preventing oxygen contaminations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Sarah Schulz reports financial support was provided by the German Federal Environmental Foundation, Germany. Largus T Angenent reports financial support was provided by the Alexander von Humboldt Foundation, Germany. Largus T. Angenent reports financial support was provided by the Novo Nordisk Foundation CO(2) Research Center, Denmark. Largus T. Angenent reports financial support was provided by German Research Foundation, Germany. Bastian Molitor reports financial support was provided by the German Research Foundation, Germany., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Current food production practices contribute significantly to climate change. To transition into a sustainable future, a combination of new food habits and a radical food production innovation must occur. Single-cell protein from microbial fermentation can profoundly impact sustainability. This review paper explores opportunities offered by gas fermentation to completely replace our reliance on fossil fuels for the production of food. Together with synthetic biology, designed microbial proteins from gas fermentation have the potential to reduce our dependence on fossil fuels and make food production more sustainable., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

For Clostridium ljungdahlii , the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: (1) the RNF complex-encoding gene cluster rnfCDGEAB ; (2) the putative RNF regulator gene rseC ; and (3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC -deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB - and rseC -deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy, and in addition, contribute to understand the connection of nitrate reduction to energy metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Klask, Jäger, Casini, Angenent and Molitor.)

Methanogenic archaea of the order Methanobacteriales are widespread in anaerobic environments and play pivotal roles in microbial communities. The family of Methanobacteriaceae encompasses mesophilic and thermophilic hydrogenotrophic species. Mesophilic species are found in various natural and anthropogenic environments (e.g., are associated with the microbiome in animals and humans). Thermophilic species can be found in thermally active bogs and warm sulfuric springs, but also in anthropogenic environments, such as wastewater treatment plants and anaerobic digesters. Recently, genetic tools for Methanothermobacter thermautotrophicus ΔH, as the first representative of this order of methanogenic archaea, were successfully implemented. This protocol describes the methods for interdomain conjugational DNA transfer from Escherichia coli to M. thermautotrophicus ΔH with shuttle-vector plasmid DNA, which allows the genetic manipulation of this microbe, and provides a basis for the development of further genetic methods for this and potentially other representatives of Methanobacteriales., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene ( bgaB ) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus , we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Weatherization of residential homes is a widespread procedure to retrofit older homes to improve the energy efficiency by reducing building leakage. Several studies have evaluated the effect of weatherization on indoor pollutants, such as formaldehyde, radon, and indoor particulates, but few studies have evaluated the effect of weatherization on indoor microbial exposure. Here, we monitored indoor pollutants and bacterial communities during reductions in building leakage for weatherized single-family residential homes in New York State and compared the data to non-weatherized homes. Nine weatherized and eleven non-weatherized single-family homes in Tompkins County, New York were sampled twice: before and after the weatherization procedures for case homes, and at least 3 months apart for control homes that were not weatherized. We found that weatherization efforts led to a significant increase in radon levels, a shift in indoor microbial community, and a warmer and less humid indoor environment. In addition, we found that changes in indoor airborne bacterial load after weatherization were more sensitive to shifts in season, whereas indoor radon levels were more sensitive to ventilation rates., Competing Interests: The authors have declared that no competing interests exist.

Medium-chain carboxylic acids (MCCAs), which can be generated from organic waste and agro-industrial side streams through microbial chain elongation, are valuable chemicals with numerous industrial applications. Membrane-based liquid-liquid extraction (pertraction) as a downstream separation process to extract MCCAs has been applied successfully. Here, a novel pertraction system with submerged hollow-fiber membranes in the fermentation bioreactor was applied to increase the MCCA extraction rate and reduce the footprint. The highest average surface-corrected MCCA extraction rate of 655.2 ± 86.4 mmol C m -2  d -1 was obtained, which was higher than any other previous reports, albeit the relatively small surface area removed only 11.6% of the introduced carbon via pertraction. This submerged extraction system was able to continuously extract MCCAs with a high extraction rate for more than 8 months. The average extraction rate of MCCA by internal membrane was 3.0- to 4.7-fold higher than the external pertraction (traditional pertraction) in the same bioreactor. A broth upflow velocity of 7.6 m h -1 was more efficient to extract MCCAs when compared to periodic biogas recirculation operation as a means to prevent membrane fouling. An even higher broth upflow velocity of 40.5 m h -1 resulted in a significant increase in methane production, losing more than 30% of carbon conversion to methane due to a loss of H 2 , and a subsequent drop in the H 2 partial pressure. This resulted in the shift from a microbial community with chain elongators as the key functional group to methanogens, because the drop in H 2 partial pressure led to thermodynamic conditions that oxidizes ethanol and carboxylic acids to acetate and H 2 with methanogens as the syntrophic partner. Thus, operators of chain elongating systems should monitor the H 2 partial pressure when changes in operating conditions are made., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xu, Bian, Angenent and Saikaly.)

Northern peatlands are experiencing more frequent and severe fire events as a result of changing climate conditions. Recent studies show that such a fire-regime change imposes a direct climate-warming impact by emitting large amounts of carbon into the atmosphere. However, the fires also convert parts of the burnt biomass into pyrogenic carbon. Here, we show a potential climate-cooling impact induced by fire-derived pyrogenic carbon in laboratory incubations. We found that the accumulation of pyrogenic carbon reduced post-fire methane production from warm (32 °C) incubated peatland soils by 13-24%. The redox-cycling, capacitive, and conductive electron transfer mechanisms in pyrogenic carbon functioned as an electron snorkel, which facilitated extracellular electron transfer and stimulated soil alternative microbial respiration to suppress methane production. Our results highlight an important, but overlooked, function of pyrogenic carbon in neutralizing forest fire emissions and call for its consideration in the global carbon budget estimation.

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 β-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n -butyrate, n -caproate, biomass, and H 2 /CO 2 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Esquivel-Elizondo, Bağcı, Temovska, Jeon, Bessarab, Williams, Huson and Angenent.)

Medium-chain carboxylic acids (MCCAs) are valuable platform chemicals and can be produced from waste biomass sources or syngas fermentation effluent through microbial chain elongation. We have previously demonstrated successful approaches to separate >90% purity oil with different MCCAs (MCCA oil) by integrating the anaerobic bioprocess with membrane-based liquid-liquid extraction (pertraction) and membrane electrolysis. However, two-compartment membrane electrolysis unit without pertraction was not able to separate MCCA oil. Therefore, we developed a five-compartment electrodialysis/phase separation cell (ED/PS). First, we tested an ED/PS cell in series with pertraction and achieved a maximum MCCA-oil flux of 1.7 × 10 3 g d -1 per projected area (m 2 ) (19 mL oil d -1 ) and MCCA-oil transfer efficiency [100% × moles MCCA-oil moles electrons -1 ] of 74% at 15 A m -2 . This extraction system at 15 A m -2 demonstrated a ∼10 times lower electric-power consumption (1.1 kWh kg -1 MCCA oil) than membrane electrolysis in series with pertraction (9.9 kWh kg -1 MCCA oil). Second, we evaluated our ED/PS as a stand-alone unit when integrated with the anaerobic bioprocess and demonstrated that we can selectively extract and separate MCCA oil directly from chain-elongating bioreactor broth with just an abiotic electrochemical cell. However, the electric-power consumption increased considerably due to the lower MCCA concentrations in the bioreactor broth compared to the pertraction broth.

The mechanism of long-distance electron transfer via redox-active particulate natural organic matter (NOM) is still unclear, especially considering its aggregated nature and the resulting low diffusivity of its quinone- and hydroquinone-containing molecules. Here we conducted microbial iron(III) mineral reduction experiments in which anthraquinone-2,6-disulfonate (AQDS, a widely used analogue for quinone- and hydroquinone-containing molecules in NOM) was immobilized in agar to achieve a spatial separation between the iron-reducing bacteria and ferrihydrite mineral. Immobilizing AQDS in agar also limited its diffusion, which resembled electron-transfer behavior of quinone- and hydroquinone-containing molecules in particulate NOM. We found that, although the diffusion coefficient of the immobilized AQDS/AH 2 QDS was 10 times lower in agar than in water, the iron(III) mineral reduction rate (1.60 ± 0.28 mmol L - 1 Fe(II) d -1 ) was still comparable in both media, indicating the existence of another mechanism that accelerated the electron transfer under low diffusive conditions. We found the correlation between the heterogeneous electron-transfer rate constant (10 -3 cm s -1 ) and the diffusion coefficient (10 -7 cm 2 s -1 ) fitting well with the "diffusion-electron hopping" model, suggesting that electron transfer via the immobilized AQDS/AH 2 QDS couple was accomplished through a combination of diffusion and electron hopping. Electron hopping increased the diffusion concentration gradient up to 10 6 -fold, which largely promoted the overall electron-transfer rate during microbial iron(III) mineral reduction. Our results are helpful to explain the electron-transfer mechanisms in particulate NOM.

Acetogenic bacteria are rising in popularity as chassis microbes for biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii . The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.

The biochemical methane potential (BMP) assay is a standard method for characterizing biomethane potential and anaerobic biodegradability organic waste streams. Therefore, the BMP protocol must be standardized to reliably compare these parameters for various substrates. Here, the effect of inoculum selection on biomethane potential was investigated through BMP tests using two different substrates and inocula obtained from four different anaerobic digesters. It was found that inocula in the form of granular sludge yielded overall higher biomethane potential and generally had faster kinetics than suspended biomass. Furthermore, acclimation of inocula to substrate appeared to have little effect on degradation rates, and co-inoculation (with both suspended and granular biomass) did not perform better than single inoculation (e.g., with suspended sludge alone). These results emphasize the role of granular sludge as an preferable inoculum for BMP assay., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The pH-value in fermentation broth is a critical factor for the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiments. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be achieved in bioreactors. Here, we present a self-built, comparatively cheap, and user-friendly multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. We tested the functionality of the MBS by cultivating the acetogen Clostridium ljungdahlii with CO 2 and H 2 at steady-state conditions (=chemostat). The experiments (total of 10 bioreactors) were addressing the two questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; and (2) does feeding nitrate influence the product spectrum under controlled pH conditions with CO 2 and H 2 ? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. On the one hand, our data showed high reproducibility for gas-fermentation experiments with C. ljungdahlii under standard cultivation conditions using the MBS. On the other hand, feeding nitrate as sole N-source improved growth by up to 62% and ethanol production by 2-3-fold. However, we observed differences in growth, and acetate and ethanol production rates between all nitrate bioreactors. We explained the different performances with a pH-buffering effect that resulted from the interplay between undissociated acetic acid production and ammonium production and because of stochastic inhibition events, which led to complete crashes at different operating times., (Copyright © 2020 Klask, Kliem-Kuster, Molitor and Angenent.)

Redox-active organic molecules such as anthraquinone-2,6-disulfonate (AQDS) and natural organic matter (NOM) can act as electron shuttles thus facilitating electron transfer from Fe(III)-reducing bacteria (FeRB) to terminal electron acceptors such as Fe(III) minerals. In this research, we examined the length scale over which this electron shuttling can occur. We present results from agar-solidified experimental incubations, containing either AQDS or NOM, where FeRB were physically separated from ferrihydrite or goethite by 2 cm. Iron speciation and concentration measurements coupled to a diffusion-reaction model highlighted clearly Fe(III) reduction in the presence of electron shuttles, independent of the type of FeRB. Based on our fitted model, the rate of ferrihydrite reduction increased from 0.07 to 0.19 μmol d -1 with a 10-fold increase in the AQDS concentration, highlighting a dependence of the reduction rate on the electron-shuttle concentration. To capture the kinetics of Fe(II) production, the effective AQDS diffusion coefficient had to be increased by a factor of 9.4. Thus, we postulate that the 2 cm electron transfer was enabled by a combination of AQDS molecular diffusion and an electron hopping contribution from reduced to oxidized AQDS molecules. Our results demonstrate that AQDS and NOM can drive microbial Fe(III) reduction across 2 cm distances and shed light on the electron transfer process in natural anoxic environments.

Microbial Fe(III) reduction plays an important role for biogeochemical carbon and iron cycling in sediments and soils. Biochar is used as a soil amendment to increase fertility and lower N 2 O/CO 2 emissions. It is redox-active and can stimulate microbial Fe(III) mineral reduction. It is currently unknown, however, how the aggregation of cells and Fe(III) minerals with biochar particles influence microbial Fe(III) reduction. Therefore, we determined rates and extent of ferrihydrite (Fh) reduction in S. oneidensis MR-1 cell suspensions with different particles sizes of wood-derived Swiss biochar and KonTiki biochar at different biochar/Fh ratios. We found that at small biochar particle size and high biochar/Fh ratios, the biochar, MR-1 cells and Fh closely aggregated, therefore addition of biochar stimulated electron transfer and microbial Fh reduction. In contrast, large biochar particles and low biochar/Fh ratios inhibited the electron transfer and Fe(III) reduction due to the lack of effective aggregation. These results suggest that for stimulating Fh reduction, a certain biochar particle size and biochar/Fh ratio is necessary leading to a close aggregation of all phases. This aggregation favors electron transfer from cells to Fh via redox cycling of the electron donating and accepting functional groups of biochar and via direct electron transfer through conductive biochar carbon matrices. These findings improve our understanding of electron transfer between microorganisms and Fe(III) minerals via redox-active biochar and help to evaluate the impact of biochar on electron transfer processes in the environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Across human populations, 16S rRNA gene-based surveys of gut microbiomes have revealed that the bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae cooccur and are enriched in individuals with a lean, compared to an obese, body mass index (BMI). Whether these association patterns reflect interactions between metabolic partners, as well as whether these associations play a role in the lean host phenotype with which they associate, remains to be ascertained. Here, we validated previously reported cooccurrence patterns of the two families and their association with a lean BMI with a meta-analysis of 1,821 metagenomes derived from 10 independent studies. Furthermore, we report positive associations at the genus and species levels between Christensenella spp. and Methanobrevibacter smithii , the most abundant methanogen of the human gut. By coculturing three Christensenella spp. with M. smithii , we show that Christensenella spp. efficiently support the metabolism of M. smithii via H 2 production far better than Bacteroides thetaiotaomicron does. Christensenella minuta forms flocs colonized by M. smithii even when H 2 is in excess. In culture with C. minuta , H 2 consumption by M. smithii shifts the metabolic output of C. minuta 's fermentation toward acetate rather than butyrate. Together, these results indicate that the widespread cooccurrence of these microorganisms is underpinned by both physical and metabolic interactions. Their combined metabolic activity may provide insights into their association with a lean host BMI. IMPORTANCE The human gut microbiome is made of trillions of microbial cells, most of which are Bacteria , with a subset of Archaea The bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae are widespread in human guts. They correlate with each other and with a lean body type. Whether species of these two families interact and how they affect the body type are unanswered questions. Here, we show that species within these families correlate with each other across people. We also demonstrate that particular species of these two families grow together in dense flocs, wherein the bacteria provide hydrogen gas to the archaea, which then make methane. When the archaea are present, the ratio of bacterial products (which are nutrients for humans) is changed. These observations indicate that when these species grow together, their products have the potential to affect the physiology of their human host., (Copyright © 2020 Ruaud et al.)

Ascaris eggs are commonly used as indicators for pathogen inactivation during the treatment of fecal sludge and wastewater due to their highly resistant lipid membrane and ability to survive in the environment for long periods of time. Current guidelines suggest that thermal treatment alone cannot inactivate Ascaris eggs at temperatures below 45 °C, although some evidence in the literature suggests this to be incorrect. Here, we performed a controlled experiment to test the effect of mesophilic temperatures on Ascaris inactivation. We exposed Ascaris suum eggs to a temperature gradient between 34°C and 45 °C under anaerobic and aerobic conditions to observe the required exposure times for a 3-log reduction. Indeed, we found that temperatures lower than 45 °C did inactivate these eggs, and the required exposure times were up to two orders of magnitude shorter than suggested by current guidelines. Results from the anaerobic exposures were used to develop a time-temperature relationship that is appropriate for Ascaris inactivation at mesophilic temperatures. Data from the literature demonstrated that our relationship is conservative, with faster inactivation occurring under environmental conditions when Ascaris eggs were suspended in fecal sludge or manure. A specific aerobic relationship was not developed, but we demonstrated that aerobic conditions cause faster inactivation than anaerobic conditions. Therefore, the anaerobic relationship provides a conservative guideline for both conditions. We demonstrate that relatively low temperatures can considerably impact Ascaris viability and suggest that mesophilic temperatures can be used in waste treatment processes to inactivate pathogens. The development of safe, low-input, mesophilic treatment processes is particularly valuable for ensuring universal access to safe sanitation and excreta management., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Anaerobic digestion coupled with combined heat and power production on dairy farms is environmentally advantageous; however, high capital and operating costs have limited its adoption, especially in the United States, where renewable electricity and heat production are under-incentivized. Biogas is also at a disadvantage because it has to compete with very low natural gas prices. The objective of this study was to evaluate the feasibility of integrating absorption refrigeration technology for combined cooling, heat, and power (CCHP) on the farm to help bridge this economic hurdle. A combined environmental life cycle and techno-economic assessment was used to compare 2 cooling pathways with and without co-digestion. We considered using CCHP to (1) displace electricity-driven refrigeration processes (e.g., milk chilling/refrigeration, biogas inlet cooling) or (2) mitigate heat stress in dairy cattle via conductive cow cooling. All cooling scenarios reduced environmental emissions compared with combined heat and power only, with an appreciable reduction in land use impacts when employing conductive cow cooling. However, none of the cooling scenarios achieved economically viability. When using cooling power to displace electricity-driven refrigeration processes, economic viability was constrained by low electricity prices and a lack of incentives in the United States. When used for conductive cow cooling, economic viability was constrained by (1) low waste heat-to-cooling conversion efficiency; (2) limited conductive cow cooling effectiveness (i.e., heat-stress mitigation); and (3) low heat stress frequency and limited severity. However, we predict that with minor improvements in conductive cow cooling effectiveness and in hotter climates, CCHP for conductive cow cooling would be economically viable even in current US energy markets., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Syntrophic microbial partnerships are found in many environments and play critical roles in wastewater treatment, global nutrient cycles, and gut systems. An important type of syntrophy for the anaerobic conversion of carboxylic acids is H 2 syntrophy. In this type of microbial partnership, dissolved H 2 is produced by a bacterium and rapidly consumed by an archeon (methanogen), resulting in methane gas. This is referred to as interspecies H 2 transfer, and some conversions rely on this mechanism to become thermodynamically feasible. For this reason, syntrophic partners are often not possible to separate in the lab, which hampers the full understanding of their physiology. Bioelectrochemical systems (BESs) may show promise to ultimately separate and study the behavior of the syntrophic bacterium by employing an abiotic H 2 oxidation reaction at the anode, actively removing dissolved H 2 . Here, we performed a proof-of-concept study to ascertain whether an H 2 -removing anode can: (1) provide a growth advantage for the syntrophic bacterium; and (2) compete with the methanogenic partner. A mathematical model was developed to design a BES to perform competition experiments. Indeed, the operated BES demonstrated the ability to provide a growth advantage to the syntrophic bacterium Syntrophus aciditrophicus compared to its methanogenic partner Methanospirillum hungatei when grown in co-culture. Further, the BES provided the never-before isolated Syntrophomonas zehnderi with a growth advantage compared to Methanobacterium formicicum . Our results demonstrate a potential to use this BES to enrich H 2 -sensitive syntrophic bacteria, and gives prospects for the development of an effective method for the separation of obligate syntrophs.

A previous study had reported that the Fenton reaction at full scale increased the digestibility of thickened sludge in a digester. The authors of the study had observed a positive effect on biogas productivity, but without a control. Here, we evaluated this result by investigating the anaerobic treatment characteristics of fresh, thickened sludge in an experimental design with a control. To accomplish this, two identical continuously stirred anaerobic digesters (CSADs) were operated in parallel at mesophilic conditions. We also included anaerobic settlers to mimic the full-scale plant and to accomplish sludge recycling. We fed fresh, thickened sludge to both setups once every other day, but performed the Fenton reaction with only the experimental system by adding H 2 O 2 to the recycled biosolids from the anaerobic settler. We observed very large fluctuations in biogas production due to ever-changing characteristics of the thickened sludge both on a daily and seasonal basis. Regardless, the two setups performed almost identically with: 1) chemical oxygen demand removal efficiencies of 63.8 ± 2.9% and 62.1 ± 3.2%; and 2) biogas productivities of 0.280 and 0.279 L CH 4 ·g -1 volatile solids for the experimental (with Fenton) and control (without Fenton) CSADs, respectively. These results indicate that the use of a Fenton reaction did not affect biogas productivities.

Syngas fermentation to ethanol and acetate has recently been coupled to microbial chain elongation to produce medium-chain carboxylates, including n-caproate and n-caprylate. These medium-chain carboxylates are relatively hydrophobic, and thus easier to extract from solution than miscible ethanol. Here, we examined the effect of 11 different ethanol-to-acetate substrate ratios (ranging from 1.8 to 275 g COD g COD -1 [1.2 to 183 mol mol -1 ]) on directing chain elongation toward n-caprylate in a 0.7-L upflow anaerobic filter with product extraction. During an eight-month operating period, we monitored the performance and characterized the microbiome composition of this chain-elongating bioreactor. We also developed a thermodynamic model to predict the favorability of n-caprylate production at different substrate ratios. As predicted by our model, higher ethanol-to-acetate substrate ratios fed to our bioreactor led to higher specificities for n-caprylate production. We observed that feeding primarily ethanol to the bioreactor (i.e., ethanol-to-acetate substrate ratio of 275 g COD g COD -1 ) resulted in the highest specificity for n-caprylate, but the n-caprylate production rate decreased at this high ratio, resulting in lower conversion efficiencies. Thus, care should be taken not to overload the system with primarily ethanol as the substrate and to lower the organic loading rate.

Pyrogenic carbon contains redox-active functional groups and polyaromatic carbon matrices that are both capable of transferring electrons. Several techniques have been explored to characterize the individual electron transfer process of either functional groups or carbon matrices individually. However, simultaneous analysis of both processes remains challenging. Using an approach that employs a four-electrode configuration and dual-interface electron transfer detection, we distinguished the electron transfer by functional groups from the electron transfer by carbon matrices and simultaneously quantified their relative contribution to the total electron transfer to and from pyrogenic carbon. Results show that at low to intermediate pyrolysis temperatures (400-500 °C), redox cycling of functional groups is the major mechanism with a contribution of 100-78% to the total electron transfer; whereas at high temperatures (650-800 °C), direct electron transfer of carbon matrices dominates electron transfer with a contribution of 87-100%. Spectroscopic and diffraction analyses of pyrogenic carbon support the electrochemical measurements by showing a molecular-level structural transition from an enrichment in functional groups to an enrichment in nanosized graphene domains with increasing pyrolysis temperatures. The method described in this study provides a new analytical approach to separately quantify the relative importance of different electron transfer pathways in natural pyrogenic carbon and has potential applications for engineered carbon materials such as graphene oxides.

The antibiotic monensin is fed to dairy cows to increase milk production efficiency. A fraction of this monensin is excreted into the cow manure. Previous studies have found that cow manure containing monensin can negatively impact the performance of anaerobic digesters, especially upon first introduction. Few studies have examined whether the anaerobic digester microbiome can adapt to monensin during the operating time. Here, we conducted a long-term time series study of four lab-scale anaerobic digesters fed with cow manure. We examined changes in both the microbiome composition and function of the anaerobic digesters when subjected to the dairy antibiotic monensin. In our digesters, monensin was not rapidly degraded under anaerobic conditions. The two anaerobic digesters that were subjected to manure from monensin feed-dosed cows exhibited relatively small changes in microbiome composition and function due to relatively low monensin concentrations. At higher concentrations of monensin, which we dosed directly to control manure (from dairy cows without monensin), we observed major changes in the microbiome composition and function of two anaerobic digesters. A rapid introduction of monensin to one of these anaerobic digesters led to the impairment of methane production. Conversely, more gradual additions of the same concentrations of monensin to the other anaerobic digester led to the adaptation of the anaerobic digester microbiomes to the relatively high monensin concentrations. A member of the candidate OP11 ( Microgenomates ) phylum arose in this anaerobic digester and appeared to be redundant with certain Bacteroidetes phylum members, which previously were dominating. IMPORTANCE Monensin is a common antibiotic given to dairy cows in the United States and is partly excreted with dairy manure. An improved understanding of how monensin affects the anaerobic digester microbiome composition and function is important to prevent process failure for farm-based anaerobic digesters. This time series study demonstrates how anaerobic digester microbiomes are inert to low monensin concentrations and can adapt to relatively high monensin concentrations by redundancy in an already existing population. Therefore, our work provides further insight into the importance of microbiome redundancy in maintaining the stability of anaerobic digesters., (Copyright © 2018 Spirito et al.)

Anaerobic digestion (AD) is a mature biotechnology-production platform with millions of installations at homes, farms, and industrial/municipal settings. Large-scale industrial, agricultural, and municipal waste-treatment systems may observe novel integration with electrochemical, biological, physical, and thermochemical process units to make AD more attractive. Without governmental subsidies, AD has often only a relatively low economic return or none at all. Diversification of products besides methane in biogas may help to change this. Here, several sections discuss different process units to: 1) upgrade biogas into biomethane; 2) convert carbon dioxide in biogas to more biomethane; 3) generate cooling power from process heat; 4) produce bio-crude oil (bio-oil) from organic matter; and 5) produce a liquid biochemical product from organic matter. This is not meant to be an exhaustive list, but rather a selection of particularly promising process units from a technological view, which are already integrated with AD or close to full-scale integration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The fermentation of synthesis gas (including carbon monoxide, carbon dioxide, and hydrogen) with anaerobic acetogens is an established biotechnological process that has recently been transferred to a commercial scale. The natural product spectrum of acetogens is natively restricted to acetate, ethanol, and 2,3-butanediol but is rapidly expanding to heterologous products. Syngas fermentation can achieve high carbon-efficiencies; however, the underlying metabolism is operating at a thermodynamic limit. This necessitates special enzymatic properties for energy conservation by acetogens. Therefore, the availability of cellular energy is considered to restrain the efficient production of energy-intense products with complex production pathways. The optimization of the feed-gas composition and other process parameters, genetic engineering, and integration with other biotechnologies is required to overcome this limitation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Discovering new ways to inactivate pathogens in human waste is critical for the improvement of worldwide access to sanitation and for the reduction of the environmental impact of conventional waste treatment processes. Here, we utilized the carboxylate platform and chain elongation to produce n-butyric acid and n-caproic acid via the anaerobic fermentation of human fecal material. Then, we inactivated Ascaris eggs through exposure to these carboxylic acids. Using batch experiments with human fecal material as substrate, we accumulated n-butyric acid and n-caproic acid at total concentrations (uncharged acid plus conjugate base) of 257 and 27.1 mM, respectively. We then showed that carboxylic acids at these concentrations inactivated Ascaris eggs when the pH was below the pK a for the acids, causing them to exist primarily in the uncharged forms. We observed that uncharged carboxylic acids affected viability rather than the pH itself or conjugate bases. In addition, we modeled the viability of Ascaris eggs as a function of uncharged carboxylic acid concentration for n-butyric acid and n-caproic acid at exposure times of 2, 6, 12, and 20 days. The results presented here indicate that in situ biological production of carboxylic acids in HFM provides a promising method of pathogen inactivation and may lead to new developments in sanitation technology and treatment of fecal sludge.

Background: Rhodopseudomonas palustris is a versatile microbe that encounters an innate redox imbalance while growing photoheterotrophically with reduced substrates. The resulting excess in reducing equivalents, together with ATP from photosynthesis, could be utilized to drive a wide range of bioconversions. The objective of this study was to genetically modify R. palustris to provide a pathway to reduce n -butyrate into n -butanol for maintaining redox balance., Results: Here, we constructed and expressed a plasmid-based pathway for n -butanol production from Clostridium acetobutylicum ATCC 824 in R. palustris. We maintained the environmental conditions in such a way that this pathway functioned as the obligate route to re-oxidize excess reducing equivalents, resulting in an innate selection pressure. The engineered strain of R. palustris grew under otherwise restrictive redox conditions and achieved concentrations of 1.5 mM n -butanol at a production rate of 0.03 g L -1  day -1 and a selectivity (i.e., products compared to the consumed substrate) of close to 40%. Since the theoretical maximum selectivity is 45%, the engineered strain converted close to its maximum selectivity., Conclusions: The innate redox imbalance of R. palustris can be used to drive the reduction of n -butyrate into n -butanol after expression of a plasmid-based enzyme from a butanol-producing Clostridium strain.

Hydrothermal liquefaction converts food waste into oil and a carbon-rich hydrothermal aqueous phase. The hydrothermal aqueous phase may be converted to biomethane via anaerobic digestion. Here, the feasibility of coupling hydrothermal liquefaction and anaerobic digestion for the conversion of food waste into energy products was examined. A mixture of polysaccharides, proteins, and lipids, representing food waste, underwent hydrothermal processing at temperatures ranging from 200 to 350°C. The anaerobic biodegradability of the hydrothermal aqueous phase was examined through conducting biochemical methane potential assays. The results demonstrate that the anaerobic biodegradability of the hydrothermal aqueous phase was lower when the temperature of hydrothermal processing increased. The chemical composition of the hydrothermal aqueous phase affected the anaerobic biodegradability. However, no inhibition of biodegradation was observed for most samples. Combining hydrothermal and anaerobic digestion may, therefore, yield a higher energetic return by converting the feedstock into oil and biomethane., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Autotrophic microbes can acquire electrons from solid donors such as steel, other microbial cells, or electrodes. Based on this feature, bioprocesses are being developed for the microbial electrosynthesis (MES) of useful products from the greenhouse gas CO 2 . Extracellular electron-transfer mechanisms involved in the acquisition of electrons from metals by electrical microbially influenced corrosion (EMIC), from other living cells by interspecies electron transfer (IET), or from an electrode during MES rely on: (i) mediators such as H 2 ; (ii) physical contact through electron-transfer proteins; or (iii) mediator-generating enzymes detached from cells. This review explores the interactions of autotrophs with solid electron donors and their importance in nature and for biosustainable technologies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Surface functional groups constitute major electroactive components in pyrogenic carbon. However, the electrochemical properties of pyrogenic carbon matrices and the kinetic preference of functional groups or carbon matrices for electron transfer remain unknown. Here we show that environmentally relevant pyrogenic carbon with average H/C and O/C ratios of less than 0.35 and 0.09 can directly transfer electrons more than three times faster than the charging and discharging cycles of surface functional groups and have a 1.5 V potential range for biogeochemical reactions that invoke electron transfer processes. Surface functional groups contribute to the overall electron flux of pyrogenic carbon to a lesser extent with greater pyrolysis temperature due to lower charging and discharging capacities, although the charging and discharging kinetics remain unchanged. This study could spur the development of a new generation of biogeochemical electron flux models that focus on the bacteria-carbon-mineral conductive network.

Background: The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures., Results: Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n -caproic acid, which were up to 40 mM day -1 (4.64 g L -1  day -1 ) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n -caproic acid production. In addition, n -caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions., Conclusions: Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.

Diseases that favor colonization of the respiratory tract with Pseudomonas aeruginosa are characterized by an altered airway microbiome. Virulence of P. aeruginosa respiratory tract infection is likely influenced by interactions with other lung microbiota or their products. The bacterial fermentation product 2,3-butanediol enhances virulence and biofilm formation of P. aeruginosa in vitro. This study assessed the effects of 2,3-butanediol on P. aeruginosa persistence, inflammatory response, and the lung microbiome in vivo. Here, P. aeruginosa grown in the presence of 2,3-butanediol and encapsulated in agar beads persisted longer in the murine respiratory tract, induced enhanced TNF-α and IL-6 responses and resulted in increased colonization in the lung tissue by environmental microbes. These results led to the following hypothesis that now needs to be tested with a larger study: fermentation products from the lung microbiota not only have a role in P. aeruginosa virulence and abundance, but also on the increased colonization of the respiratory tract with environmental microbes, resulting in dynamic shifts in microbiota diversity and disease susceptibility.

To convert wastes into sustainable liquid fuels and chemicals, new resource recovery technologies are required. Chain elongation is a carboxylate-platform bioprocess that converts short-chain carboxylates (SCCs) (e.g., acetate [C2] and n-butyrate [C4]) into medium-chain carboxylates (MCCs) (e.g., n-caprylate [C8] and n-caproate [C6]) with hydrogen gas as a side product. Ethanol or another electron donor (e.g., lactate, carbohydrate) is required. Competitive MCC productivities, yields (product vs. substrate fed), and specificities (product vs. all products) were only achieved previously from an organic waste material when exogenous ethanol had been added. Here, we converted a real organic waste, which inherently contains ethanol, into MCCs with n-caprylate as the target product. We used wine lees, which consisted primarily of settled yeast cells and ethanol from wine fermentation, and produced MCCs with a reactor microbiome. We operated the bioreactor at a pH of 5.2 and with continuous in-line extraction and achieved a MCC productivity of 3.9 g COD/L-d at an organic loading rate of 5.8 g COD/L-d, resulting in a promising MCC yield of 67% and specificities of 36% for each n-caprylate and n-caproate (72% for both). Compared to all other studies that used complex organic substrates, we achieved the highest n-caprylate-to-ncaproate product ratio of 1.0 (COD basis), because we used increased broth-recycle rates through the forward membrane contactor, which improved in-line extraction rates. Increased recycle rates also allowed us to achieve the highest reported MCC production flux per membrane surface area thus far (20.1 g COD/m 2 -d). Through microbial community analyses, we determined that an operational taxonomic unit (OTU) for Bacteroides spp. was dominant and was positively correlated with increased MCC productivities. Our data also suggested that the microbiome may have been shaped for improved MCC production by the high broth-recycle rates. Comparable abiotic studies suggest that further increases in the broth-recycle rates could improve the overall mass transfer coefficient and its corresponding MCC production flux by almost 30 times beyond the maximum that we achieved. With improved in-line extraction, the chain-elongation biotechnology production platform offers new opportunities for resource recovery and sustainable production of liquid fuels and chemicals.

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.

The conventional treatment of municipal wastewater by means of activated sludge is typically energy demanding. Here, the potential benefits of: (1) the optimization of mesophilic digestion; and (2) transitioning to thermophilic sludge digestion in three wastewater treatment plants (Tilburg-Noord, Land van Cuijk and Bath) in the Netherlands is evaluated, including a full-scale trial validation in Bath. In Tilburg-Noord, thermophilic sludge digestion covered the energy requirements of the plant (102%), whereas 111% of sludge operational treatment costs could be covered in Bath. Thermophilic sludge digestion also resulted in a strong increase in nutrient release. The potential for nutrient recovery was evaluated via: (1) stripping/absorption of ammonium; (2) autotrophic removal of ammonium via partial nitritation/anammox; and (3) struvite precipitation. This research shows that optimization of sludge digestion may lead to a strong increase in energy recovery, sludge treatment costs reduction, and the potential for advanced nutrient management in full-scale sewage treatment plants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c -type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production.