Biology subjects

Kleinsteuber, S.

Publications and source records attributed to Kleinsteuber, S..

8 recordsLinked to original sources

bioRxiv2025

Reduced carbon emissions and chain elongation during mixotrophic fermentation of a biomass feedstock

Anaerobic fermentation of biomass feedstocks using open cultures is a promising technology to produce platform carboxylates. Syngas, a mixture of H2, CO2, and CO, can be sourced sustainably and used to supplement biomass feedstocks as a source of acetyl-CoA, an intermediate for carboxylate chain elongation. To test this, syngas and corn silage were provided to a 10-L semi-continuous fermenter for 209 days of operation in a first-of-a-kind study at this scale. After acclimation to syngas, a two-fold reduction in average CO2 production rate (0.097 vs. 0.21 g L-1d-1) was observed over a period of 42 days in comparison to a control. Syngas co-feeding also increased average production rates of n-butyrate (C4) and n-caproate (C6) by 74% and 27%, respectively, although these effects were observed at relatively low C6 concentrations up to 4 g L-1. Relative abundances of Megasphaera and Dialister showed significant correlation (p<0.05) to consumption of H2 and CO as well as production of C4 and C6, suggesting involvement of these genera in mixotrophic metabolism. A feasibility analysis showed that syngas recirculation could return additional 1.54 USD m-3broth while costing 1.26 USD m-3broth and avoiding 1.81 kg CO2 eq. m-3broth in emissions compared to heterotrophic fermentation. We propose mixotrophic fermentation as a low-tech technology to turn fermenters into decentralized industrial carbon sinks. SYNOPSISFermentation of biomass and syngas could be used for sustainable and circular chemical production. This study tested the feasibility of this technology and identified relevant process parameters and microbial genera.

bioengineering↗

bioRxiv2024

A novel sulfatase for acesulfame degradation in wastewater treatment plants as evidenced from Shinella strains

The artificial sweetener acesulfame is a persistent pollutant in wastewater worldwide. So far, only a few bacterial isolates were recently found to degrade acesulfame efficiently. In Bosea and Chelatococcus strains, a Mn2+-dependent metallo-{beta}-lactamase-type sulfatase and an amidase signature family enzyme catalyze acesulfame hydrolysis via acetoacetamide-N-sulfonate (ANSA) to acetoacetate. Here, we describe a new acesulfame sulfatase in Shinella strains isolated from German wastewater treatment plants. Their genomes do not encode the Mn2+-dependent sulfatase. Instead, a formylglycine-dependent sulfatase gene was found, together with the ANSA amidase gene on a plasmid shared by all known acesulfame-degrading Shinella strains. Heterologous expression, shotgun proteomics and size exclusion chromatography corroborated the physiological function of the Shinella enzyme as a Mn2+-independent acesulfame sulfatase. Since both the Bosea/Chelatococcus sulfatase and the novel Shinella sulfatase are absent in other bacterial genomes or metagenome assembled genomes, we surveyed 60 tera base pairs of wastewater-associated metagenome raw datasets. The Bosea/Chelatococcus sulfatase gene was regularly found from 2014 on, particularly in North America, Europe and East Asia, whereas the Shinella sulfatase gene was first detected in 2020. The complete Shinella pathway is only present in five datasets from China, Finland and Mexico, suggesting that it emerged quite recently in wastewater treatment facilities. SynopsisA novel sulfatase was identified that hydrolyzes the once recalcitrant xenobiotic acesulfame. Surveying metagenome datasets revealed the recent emergence of gene homologs encoding this sulfatase in wastewater treatment systems worldwide.

genomics↗

bioRxiv2022

Functional redundancy secures resilience of chain elongation communities upon pH shifts in closed bioreactor ecosystems

For anaerobic mixed cultures performing microbial chain elongation, it is unclear how pH alterations affect the abundance of key players, microbial interactions and community functioning in terms of medium-chain carboxylate yields. We explored pH effects on mixed cultures enriched in continuous anaerobic bioreactors representing closed model ecosystems. Increasing the pH from 5.5 to 6.0 caused fluctuations in community composition and yields of n-butyrate, n-caproate and n-caprylate. Further pH increase to 6.5 restored previous yield values while the community entered a new state characterized by lower diversity and evenness but apparently higher richness, indicating the presence of species below the detection threshold in the previous state. We applied Aitchison PCA clustering, linear mixed-effects models and random forest classification on our datasets. Different pH preferences of two key chain elongation species - one Clostridium IV species related to Ruminococcaceae bacterium CPB6, and one Clostridium sensu stricto species related to Clostridium luticellarii - were determined. Based on relative abundances measured by 16S rRNA amplicon sequencing, network analysis revealed positive correlations of Clostridium IV with lactic acid bacteria, which switched from Olsenella to Lactobacillus along the pH increase, illustrating the plasticity of the food web in chain elongation communities. The pH increase induced dramatic shifts in community composition whereas process performance (in terms of product range and yields) returned to the previous state. Despite long-term cultivation in closed systems over the pH shift experiment, the communities retained functional redundancy in fermentation pathways, reflected by the emergence of rare species and concomitant recovery of chain elongation functions.

microbiology↗

bioRxiv2022

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and a spontaneous acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA hydrolase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and might have evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

microbiology↗

bioRxiv2022

Formate-induced CO tolerance and innovative methanogenesis inhibition in co-fermentation of syngas and plant biomass for carboxylate production

Production of monocarboxylates using microbial communities is highly dependent on local and degradable biomass feedstocks. Syngas or different mixtures of H2, CO, and CO2 can be co-fed to a fermenter to alleviate this dependence. To understand the effects of adding these gases during anaerobic fermentation of plant biomass, a series of batch experiments was carried out with different syngas compositions and corn silage (pH 6.0, 32{degrees}C). Co-fermentation of syngas with corn silage increased the overall carboxylate yield per gram of volatile solids (VS) by up to 44% (0.36 {+/-} 0.07 g gVS-1; in comparison to 0.23 {+/-} 0.04 g gVS-1 with a N2/CO2 headspace), despite slowing down biomass degradation. Ethylene and CO exerted a synergistic effect in preventing methanogenesis, leading to net carbon fixation. Less than 12% of the electrons were misrouted to CH4 when either 15 kPa CO or 5 kPa CO + 1.5 kPa ethylene was used. CO increased the selectivity to acetate and propionate, which accounted for 86% (electron equivalents) of all products at 49 kPa CO, by favoring lactic acid bacteria and actinobacteria over n-butyrate and n-caproate producers. This happened even when an inoculum pre-acclimatized to syngas and lactate was used. Intriguingly, the effect of CO on n-butyrate and n-caproate production was reversed when formate was present in the broth. The concept of co-fermenting syngas and plant biomass shows promise in two aspects: by making anaerobic fermentation a carbon-fixing process and by increasing the production of propionate and acetate. Testing the concept in a continuous process could improve selectivity to n-butyrate and n-caproate by enriching chain-elongating bacteria adapted to CO and complex biomass.

bioengineering↗

bioRxiv2022

Impact of fungal hyphae on growth and dispersal of obligate anaerobic bacteria in aerated habitats

Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes even in well-areated environments. Here, we analyzed whether and to which degree fractal hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches allowing for spore germination, growth and fermentative activity of the obligate anaerobe in an otherwise oxic environment. Hypoxic liquid films containing 80 {+/-} 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Our results suggest that fungal biomass typical in soil (<550 g g-1soil) may create anoxic microniches and enable activity as well as dispersal of obligate anaerobes near hyphae in an otherwise inhabitable environment.

microbiology↗

bioRxiv2021

Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation

Anaerobic fermentation with mixed cultures has gained momentum as a bioprocess for its promise to produce platform carboxylates from low-value biomass feedstocks. Anaerobic fermenters are net carbon emitters and their carboxylate yields are limited by electron donor availability. In a new approach to tackle these two disadvantages, we operated two bioreactors fed with acetate and lactate as a model feedstock while recirculating H2/CO2 to stimulate concomitant autotrophic activity. After 42 days of operation, hydrogenotrophic methanogenesis was predominant and ethylene ([≥]1.3 kPa) was added to one of the reactors, inhibiting methanogenesis completely and recovering net carbon fixation (0.20 g CO2 L-1 d-1). When methanogenesis was inhibited, exogenous H2 accounted for 17% of the consumed electron donors. Lactate-to-butyrate selectivity was 101% (88% in the control without ethylene) and lactate-to-caproate selectivity was 17% (2.3% in the control). Community analysis revealed that ethylene caused Methanobacterium to be washed out, giving room to acetogenic bacteria. In contrast to 2-bromoethanosulfonate, ethylene is a scalable methanogenesis inhibition strategy that did not collaterally block i-butyrate formation. By favoring the bacterial share of the community to become mixotrophic, the concept offers a way to simultaneously increase selectivity to medium-chain carboxylates and to develop a carbon-fixing chain elongation process.

bioengineering↗

bioRxiv2020

Microbial resource management for ex situ biomethanation of hydrogen at alkaline pH

Biomethanation is a promising solution to convert H2 produced from surplus electricity and CO2 to CH4 by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H2/CO2. We enriched a hydrogenotrophic microbiota on CO2 and H2 as sole carbon and energy sources, respectively, to investigate these competing reactions. Microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched as acetate was concomitantly produced along with CH4. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH4 ([≥] 97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities [≥]1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards selective production of CH4 from H2/CO2 by adapting the media composition, the reducing agent and the stirring intensity.

bioengineering↗