Laura Treu

Biogas Upgrading via Hydrogenotrophic Methanogenesis in Two-Stage Continuous Stirred Tank Reactors at Mesophilic and Thermophilic Conditions

This study proposes an innovative setup composed by two stage reactors to achieve biogas upgrading coupling the CO2 in the biogas with external H2 and subsequent conversion into CH4 by hydrogenotrophic methanogenesis. In this configuration, the biogas produced in the first reactor was transferred to the second one, where H2 was injected. This configuration was tested at both mesophilic and thermophilic conditions. After H2 addition, the produced biogas was upgraded to average CH4 content of 89% in the mesophilic reactor and 85% in the thermophilic. At thermophilic conditions, a higher efficiency of CH4 production and CO2 conversion was recorded. The consequent increase of pH did not inhibit the process indicating adaptation of microorganisms to higher pH levels. The effects of H2 on the microbial community were studied using high-throughput Illumina random sequences and full-length 16S rRNA genes extracted from the total sequences. The relative abundance of archaeal community markedly increased upon H2 addition with Methanoculleus as dominant genus. The increase of hydrogenotrophic methanogens and syntrophic Desulfovibrio and the decrease of aceticlastic methanogens indicate a H2-mediated shift toward the hydrogenotrophic pathway enhancing biogas upgrading. Moreover, Thermoanaerobacteraceae were likely involved in syntrophic acetate oxidation with hydrogenotrophic methanogens in absence of aceticlastic methanogenesis.

Deeper insight into the structure of the anaerobic digestion microbial community; the biogas microbiome database is expanded with 157 new genomes

This research aimed to better characterize the biogas microbiome by means of high throughput metagenomic sequencing and to elucidate the core microbial consortium existing in biogas reactors independently from the operational conditions. Assembly of shotgun reads followed by an established binning strategy resulted in the highest, up to now, extraction of microbial genomes involved in biogas producing systems. From the 236 extracted genome bins, it was remarkably found that the vast majority of them could only be characterized at high taxonomic levels. This result confirms that the biogas microbiome is comprised by a consortium of unknown species. A comparative analysis between the genome bins of the current study and those extracted from a previous metagenomic assembly demonstrated a similar phylogenetic distribution of the main taxa. Finally, this analysis led to the identification of a subset of common microbes that could be considered as the core essential group in biogas production.

Temperature‐dependent global gene expression in the Antarctic archaeon Methanococcoides burtonii

Methanococcoides burtonii is a member of the Archaea that was isolated from Ace Lake in Antarctica and is a valuable model for studying cold adaptation. Low temperature transcriptional regulation of global gene expression, and the arrangement of transcriptional units in cold-adapted archaea has not been studied. We developed a microarray for determining which genes are expressed in operons, and which are differentially expressed at low (4°C) or high (23°C) temperature. Approximately 55% of genes were found to be arranged in operons that range in length from 2 to 23 genes, and mRNA abundance tended to increase with operon length. Analysing microarray data previously obtained by others for Halobacterium salinarum revealed a similar correlation between operon length and mRNA abundance, suggesting that operons may play a similar role more broadly in the Archaea. More than 500 genes were differentially expressed at levels up to ≈ 24-fold. A notable feature was the upregulation of genes involved in maintaining RNA in a state suitable for translation in the cold. Comparison between microarray experiments and results previously obtained using proteomics indicates that transcriptional regulation (rather than translation) is primarily responsible for controlling gene expression in M. burtonii. In addition, certain genes (e.g. involved in ribosome structure and methanogenesis) appear to be regulated post-transcriptionally. This is one of few experimental studies describing the genome-wide distribution and regulation of operons in archaea.

Ex-situ biogas upgrading and enhancement in different reactor systems.

Biogas upgrading is envisioned as a key process for clean energy production. The current study evaluates the efficiency of different reactor configurations for ex-situ biogas upgrading and enhancement, in which externally provided hydrogen and carbon dioxide were biologically converted to methane by the action of hydrogenotrophic methanogens. The methane content in the output gas of the most efficient configuration was >98%, allowing its exploitation as substitute to natural gas. Additionally, use of digestate from biogas plants as a cost efficient method to provide all the necessary nutrients for microbial growth was successful. High-throughput 16S rRNA sequencing revealed that the microbial community was resided by novel phylotypes belonging to the uncultured order MBA08 and to Bacteroidales. Moreover, only hydrogenotrophic methanogens were identified belonging to Methanothermobacter and Methanoculleus genera. Methanothermobacter thermautotrophicus was the predominant methanogen in the biofilm formed on top of the diffuser surface in the bubble column reactor.

Microbial diversity and dynamicity of biogas reactors due to radical changes of feedstock composition

The anaerobic digestion process is often inhibited by alteration of substrates and/or organic overload. This study aimed to elucidate changes of microbial ecology in biogas reactors upon radical changes of substrates and to determine their importance to process imbalance. For this reason, continuously fed reactors were disturbed with pulses of proteins, lipids and carbohydrates and the microbial ecology of the reactors were characterized by 16S rRNA gene sequencing before and after the imposed changes. The microbial composition of the three reactors, initially similar, diverged greatly after substrate change. The greatest increase in diversity was observed in the reactor supplemented with carbohydrates and the microbial community became dominated by lactobacilli, while the lowest corresponded to the reactor overfed with proteins, where only Desulfotomaculum showed significant increase. The overall results suggest that feed composition has a decisive impact on the microbial composition of the reactors, and thereby on their performance.

The impact of genomic variability on gene expression in environmental Saccharomyces cerevisiae strains.

Environmental Saccharomyces cerevisiae strains are crucially important, as they represent the large pool from which domesticated industrial yeasts have been selected, and vineyard strains can be considered the genetic reservoir from which industrial wine strains with strong fermentative behaviour are selected. Four vineyard strains with different fermentation performances were chosen from a large collection of strains isolated from Italian vineyards. Their genomes were sequenced to identify how genetic variations influence gene expression during fermentation and to clarify the evolutionary relationship between vineyard isolates and industrial wine strains. RNA sequencing was performed on the four vineyard strains, as well as on the industrial wine yeast strain EC1118 and on the laboratory strain S288c, at two stages of fermentation. We showed that there was a large gene cluster with variable promoter regions modifying gene expression in the strains. Our results indicate that it is the evolvability of the yeast promoter regions, rather than structural variations or strain-specific genes, that is the main cause of the differences in gene expression. This promoter variability, determined by variable tandem repeats and a high number of single-nucleotide polymorphisms together with 49 differentially expressed transcription factors, explained the strong phenotypic differences in the strains.

Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members

In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a reference metagenome. As a second step, RNA reads were aligned on the genes encoded by the microbial community, revealing the expression of more than 51 000 different transcripts. The present study is the first research which was able to dissect the transcriptional behavior at a single species level by considering the 106 microbial genomes previously identified. The exploration of the metabolic pathways confirmed the importance of Syntrophomonas species in fatty acids degradation, and also highlighted the presence of protective mechanisms toward the long chain fatty acid effects in bacteria belonging to Clostridiales, Rykenellaceae, and in species of the genera Halothermothrix and Anaerobaculum. Additionally, an interesting transcriptional activation of the chemotaxis genes was evidenced in seven species belonging to Clostridia, Halothermothrix, and Tepidanaerobacter. Surprisingly, methanogens revealed a very versatile behavior different from each other, even among similar species of the Methanoculleus genus, while a strong increase of the expression level in Methanosarcina sp. was evidenced after oleate addition.

Dynamic functional characterization and phylogenetic changes due to Long Chain Fatty Acids pulses in biogas reactors

The process stability of biogas plants is often deteriorated by the accumulation of Long Chain Fatty Acids (LCFA). The microbial community shifts due to LCFA disturbances have been poorly understood as the molecular techniques used were not able to identify the genome characteristics of uncultured microorganisms, and additionally, the presence of limited number of reference genomes in public databases prevented the comprehension of specific functional roles characterizing these microorganisms. The present study is the first research which deciphers by means of high throughput shotgun sequencing the dynamics of the microbial community during an inhibitory shock load induced by single pulses of unsaturated LCFA at two different concentrations (i.e. 2 g/L-reactor and 3 g/L-reactor). The metagenomic analysis showed that only the microbes associated with LCFA degradation could encode proteins related to “chemotaxis” and “flagellar assembly”, which promoted the ability to move towards the LCFA sources so as to degrade them. Moreover, the syntrophic interactions found between Syntrophomonas sp. together with Methanosarcina sp. were possibly assigned to the menaquinone-electron transfer. Finally, it was proven that a previously exposed to LCFA inoculum is more efficient in the degradation process of LCFA due to the specialization of the microbial consortium.

Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances

We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Microbial analysis in biogas reactors suffering by foaming incidents

Foam formation can lead to total failure of digestion process in biogas plants. In the present study, possible correlation between foaming and the presence of specific microorganisms in biogas reactors was elucidated. The microbial ecology of continuous fed digesters overloaded with proteins, lipids and carbohydrates before and after foaming incidents was characterized using 16S rRNA gene sequencing. Moreover, the microbial diversity between the liquid and foaming layer was assessed. A number of genera that are known to produce biosurfactants, contain mycolic acid in their cell wall, or decrease the surface tension of the media, increased their relative abundance after foam formation. Finally, a microorganism similar to widely known foaming bacteria (Nocardia and Desulfotomaculum) was found to increase its relative abundance in all reactors once foam was observed, regardless of the used substrate. These findings suggest that foaming and specific microorganisms might have direct association which requires to be further investigated.