Microbial Interactions in Electroactive Biofilms for Environmental Engineering Applications: A Role for Nonexoelectrogens.

Microbial electrochemical systems have gained much attention over the past decade due to their potential for various environmental engineering applications ranging from energy production to wastewater treatment to bioproduction. At the heart of these systems lie exoelectrogens-microorganisms capable of exporting electrons generated during metabolism to external electron acceptors such as electrodes. The bacterial biofilm communities on these electrodes are dominated by exoelectrogens but are nonetheless extremely diverse. So far, within the field, the main focus has been on the electroactive bacteria. However, to broaden our understanding of these communities, it is crucial to clarify how the remaining inhabitants of electrode-respiring biofilms contribute to the overall function of the biofilm. Ultimately, such insights may enable improvement of microbial electrochemical systems by reshaping the community structure with naturally occurring beneficial strains.

Short-term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate.

Ribosomes are absolutely essential for growth but are, moreover, energetically costly to produce. Therefore, it is important to adjust the cellular ribosome levels according to the environmental conditions in order to obtain the highest possible growth rate while avoiding energy wastage on excess ribosome biosynthesis. Here we show, by three different methods, that the ribosomal RNA content of Escherichia coli is downregulated within minutes of the removal of an essential nutrient from the growth medium, or after transcription initiation is inhibited. The kinetics of the ribosomal RNA reduction vary depending on which nutrient the cells are starved for. The number of ribosomes per OD unit of cells is roughly halved after 80 min of starvation for isoleucine or phosphate, while the ribosome reduction is less extensive when the cells are starved for glucose. Collectively, the results presented here support the simple model proposed previously, which identifies the inactive ribosomal subunits as the substrates for degradation, since the most substantial rRNA degradation is observed under the starvation conditions that most directly affect the protein synthesis.

Differential interactions between natural clay minerals and dissolved organic matter affect reactive oxygen species formation.

Naturally occurring reactive oxygen species (ROS) are widely involved in many environmental processes. Here we investigated the ROS generation associated with the interaction between complexed natural clay minerals (CMs) and dissolved organic matter (DOM). Our results showed that among the nine chemical-reduced CMs (CR-CMs), the light brown CR-CM (CR-CM 7) generated the highest ROS via oxygenation, relying on the reactive structural Fe(II) (Fe species that can transfer electrons to oxygen) instead of total structural Fe(II) as previously reported. Moreover, DOM affected the oxygenation of CR-CMs differently. The tight interaction between DOM and CR-CM 7 formed DOM-complexed Fe, while the weak interaction between DOM and the dark gold CR-CM (CR-CM 1) and the black CR-CM (CR-CM 5) exhibited decreased efficiencies. Mechanism studies revealed that ROS were generated through three pathways but all followed a similar one-electron transfer process in the presence of DOM. We further developed a three-layer geobattery model system and demonstrated that long electron transfer driven by CR-CMs/DOM could extend ROS generation to several centimetres across the oxic-anoxic interface, even without redox switching. These findings offer new insights into CMs-involved ROS generation and associated organic matter transformation in natural environments.

Conjugative plasmids inhibit extracellular electron transfer in Geobacter sulfurreducens.

Geobacter sulfurreducens is part of a specialized group of microbes with the unique ability to exchange electrons with insoluble materials, such as iron oxides and electrodes. Therefore, G. sulfurreducens plays an essential role in the biogeochemical iron cycle and microbial electrochemical systems. In G. sulfurreducens this ability is primarily dependent on electrically conductive nanowires that link internal electron flow from metabolism to solid electron acceptors in the extracellular environment. Here we show that when carrying conjugative plasmids, which are self-transmissible plasmids that are ubiquitous in environmental bacteria, G. sulfurreducens reduces insoluble iron oxides at much slower rates. This was the case for all three conjugative plasmids tested (pKJK5, RP4 and pB10). Growth with electron acceptors that do not require expression of nanowires was, on the other hand, unaffected. Furthermore, iron oxide reduction was also inhibited in Geobacter chapellei, but not in Shewanella oneidensis where electron export is nanowire-independent. As determined by transcriptomics, presence of pKJK5 reduces transcription of several genes that have been shown to be implicated in extracellular electron transfer in G. sulfurreducens, including pilA and omcE. These results suggest that conjugative plasmids can in fact be very disadvantageous for the bacterial host by imposing specific phenotypic changes, and that these plasmids may contribute to shaping the microbial composition in electrode-respiring biofilms in microbial electrochemical reactors.

Insights into the impact of polyethylene microplastics on methane recovery from wastewater via bioelectrochemical anaerobic digestion.

Bioelectrochemical anaerobic digestion (BEAD) is a promising next-generation technology for simultaneous wastewater treatment and bioenergy recovery. While knowledge on the inhibitory effect of emerging pollutants, such as microplastics, on the conventional wastewater anaerobic digestion processes is increasing, the impact of microplastics on the BEAD process remains unknown. This study shows that methane production decreased by 30.71% when adding 10 mg/L polyethylene microplastics (PE-MP) to the BEAD systems. The morphology of anaerobic granular sludge, which was the biocatalysts in the BEAD, changed with microbes shedding and granule crack when PE-MP existed. Additionally, the presence of PE-MP shifted the microbial communities, leading to a lower diversity but higher richness and tight clustering. Moreover, fewer fermentative bacteria, acetogens, and hydrogenotrophic methanogens (BEAD enhanced) grew under PE-MP stress, suggesting that PE-MP had an inhibitory effect on the methanogenic pathways. Furthermore, the abundance of genes relevant to extracellular electron transfer (omcB and mtrC) and methanogens (hupL and mcrA) decreased. The electron transfer efficiency reduced with extracellular cytochrome c down and a lower electron transfer system activity. Finally, phylogenetic investigation of communities by reconstruction of unobserved states analysis predicted the decrease of key methanogenic enzymes, including EC 1.1.1.1 (Alcohol dehydrogenase), EC 1.2.99.5 (Formylmethanofuran dehydrogenase), and EC 2.8.4.1 (Coenzyme-B sulfoethylthiotransferase). Altogether, these results provide insight into the inhibition mechanism of microplastics in wastewater methane recovery and further optimisation of the BEAD process.

Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing.

Escherichia coli cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard E. coli K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to E. coli K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.