Elucidating the development of cooperative anode-biofilm-structures.

Microbial electrochemical systems are a highly versatile platform technology with a particular focus on the interplay of chemical and electrical energy conversion and offer immense potential for a sustainable bioeconomy. The industrial realization of this potential requires a critical focus on biofilm optimization if performance is to be controlled over a long period of time. Moreover, the aspect and influence of cooperativity has to be addressed as many applied anodic bioelectrochemical systems will most likely be operated with a diversity of interacting microbial species. Hence, the aim of this study was to analyze how interspecies dependence and cooperativity of a model community influence the development of anodic biofilms. To investigate biofilm activity in a spatially resolved manner, a microfluidic bioelectrochemical flow cell was developed that can be equipped with user-defined electrode materials and operates under laminar flow conditions. With this infrastructure, the development of single and co-culture biofilms of the two model organisms Shewanella oneidensis and Geobacter sulfurreducens on graphite electrodes was monitored by optical coherence tomography analysis. The interdependence in the co-culture biofilm was achieved by feeding the community with lactate, which is converted by S. oneidensis into acetate, which in turn serves as substrate for G. sulfurreducens. The results show that co-cultivation resulted in the formation of denser biofilms than in single culture. Moreover, we hypothesize that S. oneidensis in return utilizes the conductive biofilm matrix build by G. sulfurreducens for direct interspecies electron transfer (DIET) to the anode. FISH analysis revealed that the biofilms consisted of approximately two-thirds G. sulfurreducens cells, which most likely formed a conductive 3D network throughout the biofilm matrix, in which evenly distributed tubular S. oneidensis colonies were embedded without direct contact to the anode surface. Live/dead staining shows that the outermost biofilm contained almost exclusively dead cells (98 %), layers near the anode contained 45-56 % and the entire biofilm contained 82 % live cells. Our results exemplify how the architecture of the exoelectrogenic biofilm dynamically adapts to the respective process conditions.

Biofilms for Production of Chemicals and Energy.

The twenty-first century will be the century of biology. This is not only because of breakthrough advances in molecular biology tools but also because we need to reinvent our economy based on the biological principles of energy efficiency and sustainability. Consequently, new tools for production routines must be developed to help produce platform chemicals and energy sources based on sustainable resources. In this context, biofilm-based processes have the potential to impact future production processes, because they can be carried out continuously and with robust stationary biocatalysts embedded in an extracellular matrix with different properties. We review productive biofilm systems used for heterotrophic and lithoautotrophic production and attempt to identify fundamental reasons why they may be particularly suitable as future production systems. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering , Volume 15 is June 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Beneficial applications of biofilms.

Many microorganisms live in the form of a biofilm. Although they are feared in the medical sector, biofilms that are composed of non-pathogenic organisms can be highly beneficial in many applications, including the production of bulk and fine chemicals. Biofilm systems are natural retentostats in which the biocatalysts can adapt and optimize their metabolism to different conditions over time. The adherent nature of biofilms allows them to be used in continuous systems in which the hydraulic retention time is much shorter than the doubling time of the biocatalysts. Moreover, the resilience of organisms growing in biofilms, together with the potential of uncoupling growth from catalytic activity, offers a wide range of opportunities. The ability to work with continuous systems using a potentially self-advancing whole-cell biocatalyst is attracting interest from a range of disciplines, from applied microbiology to materials science and from bioengineering to process engineering. The field of beneficial biofilms is rapidly evolving, with an increasing number of applications being explored, and the surge in demand for sustainable and biobased solutions and processes is accelerating advances in the field. This Review provides an overview of the research topics, challenges, applications and future directions in beneficial and applied biofilm research.

Gastropods as a source for fecal indicator bacteria in drinking water.

Microbial water quality is routinely examined using the fecal indicator bacteria Escherichia coli, coliform bacteria and enterococci. Several practical cases in German drinking water distribution systems indicated invertebrates such as insects or gastropods as a source for the microbiological deterioration. Therefore, we examined three genera of Gastropoda (Arion, Helix and Cepaea) for the presence of fecal indictor bacteria in excreta using standard methods. Enterococci and coliform bacteria were detected in high concentrations (mean values of 1.5 × 106 and 6.3 × 106 per gram feces, respectively). E. coli was also detected, still specification revealed that what was assigned by standard ISO-methods to be E. coli was indeed a novel species of Buttiauxella, exhibiting ß-D-glucuronidase activity, thus, explaining the false-positive results. Microbiome analyses confirmed the cultural results. Enterobacteriaceae were dominant in the samples, yet only very few sequences could be assigned to Escherichia. Our study suggests, that enterococci and coliform bacteria are an integral component in the gastropod microbiome, whereas E. coli might be derived from other sources with gastropods being a vector. The results further indicate, that the current concept of fecal indicator bacteria needs to be extended, as not only humans and homeothermic animals could be a source for fecal indictor bacteria, but also gastropods need to be taken into consideration.

Microbe-Anode Interactions: Comparing the impact of genetic and material engineering approaches to improve the performance of microbial electrochemical systems (MES).

Microbial electrochemical systems (MESs) are a highly versatile platform technology with a particular focus on power or energy production. Often, they are used in combination with substrate conversion (e.g., wastewater treatment) and production of value-added compounds via electrode-assisted fermentation. This rapidly evolving field has seen great improvements both technically and biologically, but this interdisciplinarity sometimes hampers overseeing strategies to increase process efficiency. In this review, we first briefly summarize the terminology of the technology and outline the biological background that is essential for understanding and thus improving MES technology. Thereafter, recent research on improvements at the biofilm-electrode interface will be summarized and discussed, distinguishing between biotic and abiotic approaches. The two approaches are then compared, and resulting future directions are discussed. This mini-review therefore provides basic knowledge of MES technology and the underlying microbiology in general and reviews recent improvements at the bacteria-electrode interface.

Probing the robustness of Geobacter sulfurreducens against fermentation hydrolysate for uses in bioelectrochemical systems.

In this study, impacts of toxic ions/acids found in real fermentation-hydrolysate on the model exoelectrogenic G. sulfurreducens were investigated. Initially, different concentrations of acetate, butyrate, propionate, Na+, and K+ were tested, individually and in combination, for effects on the planktonic growth, followed by validation with diluted-hydrolysate. Meanwhile, it could be shown that (1) excess Na+ (≥100 mM) causes inhibition that can be reduced by K+ replacement, (2) butyrate (≥10 mM) induces higher toxicity than propionate, and (3) hydrolysate induces synergistic inhibition to G. sulfurreducens where organic constituents contributed more than Na+. Afterwards, compared with impacts on planktonic cells, the pre-enriched anodic biofilm of G. sulfurreducens in BESs showed higher robustness against diluted-hydrolysate, achieving current densities of 1.4-1.7 A/m2 (at up to ∼30 mM butyrate and propionate as well as ∼240 mM Na+). As a conclusion, using G. sulfurreducens in BESs dealing with fermentation-hydrolysate can be regulated for efficacious energy recovery.

Electron transfer of extremophiles in bioelectrochemical systems.

The interaction of bacteria and archaea with electrodes is a relatively new research field which spans from fundamental to applied research and influences interdisciplinary research in the fields of microbiology, biochemistry, biotechnology as well as process engineering. Although a substantial understanding of electron transfer processes between microbes and anodes and between microbes and cathodes has been achieved in mesophilic organisms, the mechanisms used by microbes under extremophilic conditions are still in the early stages of discovery. Here, we review our current knowledge on the biochemical solutions that evolved for the interaction of extremophilic organisms with electrodes. To this end, the available knowledge on pure cultures of extremophilic microorganisms has been compiled and the study has been extended with the help of bioinformatic analyses on the potential distribution of different electron transfer mechanisms in extremophilic microorganisms.

Sprayable biofilm - Agarose hydrogels as 3D matrix for enhanced productivity in bioelectrochemical systems.

Bio-based energy production utilizing renewable resources can be realized by exoelectrogenic organisms and their application in bioelectrochemical systems (BES). These organisms catalyze the direct conversion of chemical into electrical energy and are already widely used in bioelectronics and biosensing. However, the biofilm-electrode interaction is a factor that limits sufficient space-time-yields for industrial applications. In this study, a hydrogel matrix consisting of agarose fibers was utilized as a scaffold for S. oneidensis cells to improve anodic processes in BES. This synthetic, scalable biofilm reached a higher current density in BES in comparison to naturally formed biofilms. Complemented with carbon nanofibers and riboflavin, the application of this functionalized hydrogel containing S. oneidensis cells led to an overall 9.1-fold increase in current density to 1324 mA m-2 in comparison to 145 mA m-2 for the planktonic control. In addition, the synthetic biofilm can be applied by spraying onto surfaces using a novel spray applicator. The latter allows to apply the biofilm effortless on large surfaces which will facilitate scalability and thus industrial application.

Microbially promoted calcite precipitation in the pelagic redoxcline: Elucidating the formation of the turbid layer.

Large bell-shaped calcite formations called "Hells Bells" were discovered underwater in the stratified cenote El Zapote on the Yucatán Peninsula, Mexico. Together with these extraordinary speleothems, divers found a white, cloudy turbid layer into which some Hells Bells partially extend. Here, we address the central question if the formation of the turbid layer could be based on microbial activity, more specifically, on microbially induced calcite precipitation. Metagenomic and metatranscriptomic profiling of the microbial community in the turbid layer, which overlaps with the pelagic redoxcline in the cenote, revealed chemolithoautotrophic Hydrogenophilales and unclassified ß-Proteobacteria as the metabolic key players. Bioinformatic and hydrogeochemical data suggest chemolithoautotrophic oxidation of sulfide to zero-valent sulfur catalyzed by denitrifying organisms due to oxygen deficiency. Incomplete sulfide oxidation via nitrate reduction and chemolithoautotrophy are both proton-consuming processes, which increase the pH in the redoxcline favoring authigenic calcite precipitation and may contribute to Hells Bells growth. The observed mechanism of microbially induced calcite precipitation is potentially applicable to many other stagnant sulfate-rich water bodies.

The importance of biofilm formation for cultivation of a Micrarchaeon and its interactions with its Thermoplasmatales host.

Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses.

Nanowired electrodes as outer membrane cytochrome-independent electronic conduit in Shewanella oneidensis.

Extracellular electron transfer (EET) from microorganisms to inorganic electrodes is a unique ability of electrochemically active bacteria. Despite rigorous genetic and biochemical screening of the c-type cytochromes that make up the EET network, the individual electron transfer steps over the cell membrane remain mostly unresolved. As such, attempts to transplant entire EET chains from native into non-native exoelectrogens have resulted in inferior electron transfer rates. In this study we investigate how nanostructured electrodes can interface with Shewanella oneidensis to establish an alternative EET pathway. Improved biocompatibility was observed for densely packed nanostructured surfaces with a low cell-nanowire load distribution during applied external forces. External gravitational forces were needed to establish a bioelectrochemical cell-nanorod interface. Bioelectrochemical analysis showed evidence of nanorod penetration beyond the outer cell membrane of a deletion mutant lacking all outer membrane cytochrome encoding genes that was only electroactive on a nanostructured surface and under external force.

A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer.

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in "Ca. Micrarchaeum harzensis A_DKE," and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated, and the micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described coculture, containing "Ca. Micrarchaeum harzensis A_DKE" and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of "Ca. Micrarchaeum harzensis A_DKE." Both organisms are clearly distinguishable by cell size, shape, and surface structure. IMPORTANCE Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member "Candidatus Micrarchaeum harzensis A_DKE," an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of "Ca. Micrarchaeum harzensis A_DKE" is an important step toward understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime.

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon.

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as "Candidatus Micrarchaeum harzensis A_DKE", remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE's isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP+ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and kcat-values of 38.48 ± 1.62 and 43.99 ± 1.46 s-1 resulting in kcat/KM-values of 725 ± 107.62 and 22.69 ± 2.15 mM-1 s-1 for DL-isocitrate and NADP+, respectively. MhIDH's exceptionally low affinity for NADP+, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP+ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.

Developing Rhodobacter sphaeroides for cathodic biopolymer production.

In this work, Rhodobacter sphaeroides was identified as a potential cathodic production strain for photoautotrophic production processes. First, a stable cultivation in a bioelectrochemical system (BES) was established under conditions in which hydrogen produced by a poised cathode served as an electron donor. It was shown that both the introduction of a plasmid vector and exposure to the corresponding antibiotic selection pressure caused a strong improvement in both cathodic biofilm formation and electrochemical properties. A quantitative proteomic analysis identified key players in the molecular adaptation to biofilm growth on the cathodic surface. Furthermore, biofilm formation kinetics were quantified by optical coherence tomography measurements, which showed a strong tendency for biofilm formation together with a robust biofilm architecture. A media switch to N2-limited conditions resulted in increased cathodic poly(3-hydroxybutyrate) (PHB) accumulation, suggesting R. sphaeroides as a potential strain for photoautotrophic PHB production in future industrial applications.

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis.

BACKGROUND: Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles. RESULTS: In the model organism Shewanella oneidensis, riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value. CONCLUSION: This study describes a new quorum-sensing mechanism in S. oneidensis. Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC. The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems.

Biological biogas upgrading in a membrane biofilm reactor with and without organic carbon source.

Biogas upgrading is a necessary step to minimize the CO2 of raw biogas and to make it suitable for gas liquefaction or introduction into the national gas grid. Biomethanation is a promising approach since it converts the CO2 to more methane on site, while taking advantage of the organisms responsible for biogas production in the first place. This study investigates the suitability of a pseudo-dead-end membrane biofilm reactor (MBfR) for ex-situ biogas upgrading using biogas as sole carbon source as well as for additional acetoclastic methanation when an organic carbon source is provided. Results prove that the concept of MBfR is especially advantageous for ex-situ hydrogenotrophic methanation of biogas CO2, yielding high product gas qualities of up to 99% methane. It is discussed that cross-flow membrane operation could reduce mass flux of inert methane through membranes, attached biofilms, and reactor liquid, and, thus, improve methanation space time yields.

Production of acetoin from renewable resources under heterotrophic and mixotrophic conditions.

This study aimed to reveal whether Cupriavidus necator H16 is suited for the production of acetoin based on the carboxylic acids acetate, butyrate and propionate under heterotrophic and mixotrophic conditions. The chosen production strain, lacking the polyhydroxybutyrate synthases phaC1 and phaC2, was revealed to be beneficiary for autotrophic acetoin production. Proteomic analysis of the strain determined that the deletions do indeed have a significant impact on pyruvate formation and its subsequent direction towards the introduced acetoin-synthesis pathway. Moreover, the strain was tested for its ability to use typical dark fermentation products under hetero- and mixotrophic conditions. Growth with butyrate and acetate led to low efficiencies, while 46.54% ±0.78 of the added propionate was converted into acetoin. Interestingly, mixotrophic conditions led to simultaneous consumption of acetate and butyrate with the gaseous substrates and lowered efficiency. In contrast, mixotrophic propionate consumption led to diauxic behavior and high carbon efficiency of 71.2% ±0.64.

Assessing and modeling biocatalysis in field denitrification beds reveals key influencing factors for future constructions.

Environmental contamination with fertilizers is threatening biodiversity in many ecosystems due to nitrate-based eutrophication. One opportunity for a cost-efficient nitrate elimination are denitrification beds in which a microbial community thrives under anoxic conditions with polymeric plant material as a carbon and an electron source. Incoming nitrate is used as electron acceptor and reduced to molecular nitrogen. Projects realizing denitrification beds in field scale are sparse and robust data on their efficiency throughout the year mostly not available. This study analyzed the nitrate elimination efficiency and microbiology of a 216 m3 denitrification bed over the time course of more than three years. Phylogenetic as well as transcriptomic analysis revealed that the reactor contained a biofilm community growing on the surface of the wood chips and a planktonic community. Both differed in composition but their variance was affected only to a minor extend by seasonal temperature changes. Cellulose degradation was mainly conducted by the biofilm population while denitrification was mostly conducted by the planktonic community. Methanogens were detectable only to a very minor extend. Using online data from the nitrate concentration of in- and outflowing water as well as a hydrological model to predict the water inflow, it was possible to establish a process model that sufficiently describes the denitrification process. This model clearly indicates that the denitrification efficiency is mostly impacted by temperature and hydraulic retention time. It also suggests that the simple design of the denitrification bed most likely leads to different flow paths through the reactor depending on the volumetric flow rate. This study allows for the first time a robust estimation of the necessary reactor size for nitrate removal in a moderate continental climate setting. It also suggests how future denitrification beds could be improved for better performance.

Propionic acid production from food waste in batch reactors: Effect of pH, types of inoculum, and thermal pre-treatment.

In this study, lab-scale batch fermentation tests were carried out at mesophilic temperature (30 °C) to examine the influence of inoculum type, pH-value, and thermal pretreatment of substrate on propionic acid (PA) production from dog food. The selected inocula comprised a mixed bacterial culture, milk, and soft goat cheese. The batch tests were performed at pH 4, pH 6, and pH 8 for both, untreated and thermally pretreated food. Results show that the production of PA and volatile fatty acids (VFAs) in general were significantly dependent on the chosen inoculum and adjusted pH value. The maximum PA production rates and yields were determined for the cheese inoculum at pH 6 using untreated and pretreated dog food. PA concentration reached 10 gL-1and 26.5 gL-1, respectively. Our findings show that by selecting optimal process parameters, an efficient PA production from model food waste can be achieved.