Anaerobic digestion of process water from hydrothermal treatment processes: a review of inhibitors and detoxification approaches.

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.

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%.

Exclusive D-lactate-isomer production during a reactor-microbiome conversion of lactose-rich waste by controlling pH and temperature.

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.

An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter.

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.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

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.

Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide.

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.

An Interdomain Conjugation Protocol for Plasmid-DNA Transfer into Methanothermobacter thermautotrophicus ΔH.

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.

Genetic Evidence Reveals the Indispensable Role of the rseC Gene for Autotrophy and the Importance of a Functional Electron Balance for Nitrate Reduction in Clostridium ljungdahlii.

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.

Recycling carbon for sustainable protein production using gas fermentation.

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.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH.

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 ß-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 ß-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.

The short-term effect of residential home energy retrofits on indoor air quality and microbial exposure: A case-control study.

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.

Long-Term Continuous Extraction of Medium-Chain Carboxylates by Pertraction With Submerged Hollow-Fiber Membranes.

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 H2, and a subsequent drop in the H2 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 H2 partial pressure led to thermodynamic conditions that oxidizes ethanol and carboxylic acids to acetate and H2 with methanogens as the syntrophic partner. Thus, operators of chain elongating systems should monitor the H2 partial pressure when changes in operating conditions are made.

Suppressing peatland methane production by electron snorkeling through pyrogenic carbon in controlled laboratory incubations.

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.

Direct Medium-Chain Carboxylic Acid Oil Separation from a Bioreactor by an Electrodialysis/Phase Separation Cell.

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 × 103 g d-1 per projected area (m2) (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.

Electron Hopping Enables Rapid Electron Transfer between Quinone-/Hydroquinone-Containing Organic Molecules in Microbial Iron(III) Mineral Reduction.

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/AH2QDS 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 cm2 s-1) fitting well with the "diffusion-electron hopping" model, suggesting that electron transfer via the immobilized AQDS/AH2QDS couple was accomplished through a combination of diffusion and electron hopping. Electron hopping increased the diffusion concentration gradient up to 106-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.

Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination.

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.

Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H2, but Results in Stochastic Inhibition Events.

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 CO2 and H2 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 CO2 and H2? 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.

Stochasticity in microbiology: managing unpredictability to reach the Sustainable Development Goals.

Pure (single) cultures of microorganisms and mixed microbial communities (microbiomes) have been important for centuries in providing renewable energy, clean water and food products to human society and will continue to play a crucial role to pursue the Sustainable Development Goals. To use microorganisms effectively, microbial engineered processes require adequate control. Microbial communities are shaped by manageable deterministic processes, but also by stochastic processes, which can promote unforeseeable variations and adaptations. Here, we highlight the impact of stochasticity in single culture and microbiome engineering. First, we discuss the concepts and mechanisms of stochasticity in relation to microbial ecology of single cultures and microbiomes. Second, we discuss the consequences of stochasticity in relation to process performance and human health, which are reflected in key disadvantages and important opportunities. Third, we propose a suitable decision tool to deal with stochasticity in which monitoring of stochasticity and setting the boundaries of stochasticity by regulators are central aspects. Stochasticity may give rise to some risks, such as the presence of pathogens in microbiomes. We argue here that by taking the necessary precautions and through clever monitoring and interpretation, these risks can be mitigated.