Microbial Biotechnology

ABSTRACT Biogas, a mix of CO 2 , CH 4 and small proportions of other gases, is a biofuel obtained by anaerobic digestion (AD). Biogas production is often considered a black box process, as the role and dynamics of some of the microorganisms involved remain undisclosed. Previous metataxonomic studies in the frame of the MICRO4BIOGAS project ( www.micro4biogas.eu ) revealed that MBA03, an uncharacterised and uncultured bacterial taxon belonging to phylum Bacillota , was very prevalent and abundant in industrial full‐scale AD plants. Despite the efforts, this taxon has not yet been cultivated, which makes the analysis of its taxonomy, ecology and metabolism even more challenging. In the present work, 30 samples derived from anaerobic digesters were sequenced, allowing the reconstruction of 108 metagenome‐assembled genomes (MAGs) potentially belonging to MBA03. According to phylogenetic analyses and genomic similarity indices, MBA03 was classified as a new bacterial order, proposed as ‘ Candidatus Darwinibacteriales’ ord. nov., which includes ‘ Candidatus Darwinibacter acetoxidans’ gen. nov., sp. nov. of ‘ Candidatus Darwinibacteriaceae’ fam. nov., along with ‘ Candidatus Wallacebacter cryptica’ gen. nov., sp. nov. of the ‘ Candidatus Wallacebacteriaceae’ fam. nov. Ecotaxonomic studies determined that AD processes are the main ecological niche of ‘ Candidatus Darwinibacteriales’. Moreover, metabolic predictions identified Darwinibacteraceae members as putative syntrophic acetate‐oxidising bacteria (SAOB), as they encode for the reversed Wood–Ljungdahl (W–L) pathway coupled to the glycine cleavage system. This suggests that Darwinibacteraceae members could work in collaboration with hydrogenotrophic methanogenic archaea to produce methane in industrial biogas plants. Overall, our findings present ‘ Candidatus Darwinibacteriales’ as a potential key player in anaerobic digestion and pave the way towards the complete characterisation of this newly described bacterial taxon, which has not yet been cultured.

ABSTRACT Biogas reactors operating at elevated ammonia levels are commonly susceptible to process disturbances, further augmented at thermophilic temperatures. The major cause is assumed to be linked to inhibition followed by an imbalance between different functional microbial groups, centred around the last two steps of the anaerobic digestion, involving acetogens, syntrophic acetate oxidisers (SAOB) and methanogens. Acetogens are key contributors to reactor efficiency, acting as the crucial link between the hydrolysis and fermentation steps and the final methanogenesis step. Their major product is acetate, at high ammonia levels further converted by SAOB and hydrogenotrophic methanogens to biogas. Even though these functionally different processes are well recognised, less is known about the responsible organism at elevated temperature and ammonia conditions. The main aim of this study was to garner insights into the penultimate stages in three thermophilic reactors (52°C) operated under high ammonia levels (FAN 0.7–1.0 g/L; TAN 3.6–4.4 g/L). The primary objective was to identify potential acetogens and SAOBs. Metagenomic data from the three reactors were analysed for the reductive acetyl‐CoA pathway (Wood–Ljungdahl Pathway) and glycine synthase reductase pathway. The results revealed a lack of true acetogens but uncovered three potential SAOB candidates that harbour the WLP, ‘ Candidatus Thermodarwinisyntropha acetovorans’, ‘ Candidatus Thermosyntrophaceticus schinkii’, ‘ Candidatus Thermotepidanaerobacter aceticum’, and a potential lipid‐degrader ‘ Candidatus Thermosyntrophomonas ammoiaca’.

Summary Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus‐growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri , in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri . To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus‐growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP‐225, OMP‐333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP‐47 and OMP‐225 displayed specificity to the D. citri vector. The application of OMP‐225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri . The application of OMP‐333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.

Summary ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem‐limited non‐culturable α‐proteobacterium associated with citrus Huanglongbing, a highly destructive disease threatening global citrus industry. Research on CLas is challenging due to the current inability to culture CLas in vitro and the low CLas titre in citrus plant. Here, we develop a CLas enrichment system using the holoparasitic dodder plant ( Cuscuta campestris ) as an amenable host to acquire and enrich CLas from CLas‐infected citrus shoots maintained hydroponically. Forty‐eight out of fifty‐five (87%) dodder plants successfully parasitized CLas‐infected citrus shoots with detectable CLas by PCR. Among 48 dodders cultures, 30 showed two‐ to 419‐fold CLas titre increase as compared to the corresponding citrus hosts. The CLas population rapidly increased and reached the highest level in dodder tendrils at 15 days after parasitizing citrus shoot. Genome sequencing and assembly derived from CLas‐enriched dodder DNA samples generated a higher resolution than those obtained for CLas from citrus hosts. No genomic variation was detected in CLas after transmission from citrus to dodder during short‐term parasitism. Dual RNA‐Seq experiments showed similar CLas gene expression profiles in dodder and citrus samples, yet dodder samples generated a higher resolution of CLas transcriptome data. The ability of dodder to support CLas multiplication to high levels, as well as its advantage in CLas genomic and transcriptomic analyses, make it an optimal model for further studies on CLas–host interaction.

Summary ‘ Candidatus Liberibacter asiaticus ’ is a fastidious bacterium and a putative agent of citrus greening disease (a.k.a., huanglongbing, HLB), a significant agricultural disease that affects citrus fruit quality and tree health. In citrus, ‘ Ca. L. asiaticus ’ is phloem limited. Lack of culture tools to study ‘ Ca. L. asiaticus ’ complicates analysis of this important organism. To improve understanding of ‘ Ca. L. asiaticus ’–host interactions including parameters that affect ‘ Ca. L. asiaticus ’ replication, methods suitable for screening pathogen responses to physicochemical and nutritional variables are needed. We describe a leaf disc‐based culture assay that allows highly selective measurement of changes in ‘ Ca. L. asiaticus ’ DNA within plant tissue incubated under specific physicochemical and nutritional conditions. qPCR analysis targeting the hypothetical gene CD16‐00155 (strain A4) allowed selective quantification of ‘ Ca. L. asiaticus ’ DNA content within infected tissue. ‘ Ca. L. asiaticus ’ DNA replication was observed in response to glucose exclusively under microaerobic conditions, and the antibiotic amikacin further enhanced ‘ Ca. L. asiaticus ’ DNA replication. Metabolite profiling revealed a moderate impact of ‘ Ca. L. asiaticus ’ on the ability of leaf tissue to metabolize and respond to glucose.

Summary Biocathode microbial communities are proposed to catalyse a range of useful reactions. Unlike bioanodes, model biocathode organisms have not yet been successfully cultivated in isolation highlighting the need for culture‐independent approaches to characterization. Biocathode MCL ( Marinobacter , Chromatiaceae , Labrenzia ) is a microbial community proposed to couple CO 2 fixation to extracellular electron transfer and O 2 reduction. Previous metagenomic analysis of a single MCL bioelectrochemical system ( BES ) resulted in resolution of 16 bin genomes. To further resolve bin genomes and compare community composition across replicate MCL BES , we performed shotgun metagenomic and 16S rRNA gene (16S) sequencing at steady‐state current. Clustering pooled reads from replicate BES increased the number of resolved bin genomes to 20, over half of which were > 90% complete. Direct comparison of unassembled metagenomic reads and 16S operational taxonomic units ( OTU s) predicted higher community diversity than the assembled/clustered metagenome and the predicted relative abundances did not match. However, when 16S OTU s were mapped to bin genomes and genome abundance was scaled by 16S gene copy number, estimated relative abundance was more similar to metagenomic analysis. The relative abundance of the bin genome representing ‘ Ca . Tenderia electrophaga’ was correlated with increasing current, further supporting the hypothesis that this organism is the electroautotroph.

Summary A novel candidate species of the liberibacter genus, ‘ Candidatus Liberibacter brunswickensis’ ( CL br), was identified in the Australian eggplant psyllid, Acizzia solanicola . This is the first discovery of a species belonging to the liberibacter genus in Australia and the first report of a liberibacter species in the psyllid genus Acizzia . This new candidate liberibacter species has not been associated with plant disease, unlike other psyllid‐vectored species in the genus including ‘ Candidatus Liberibacter asiaticus ’ ( CL as), ‘ Candidatus Liberibacter africanus’ ( CL af) and ‘ Ca . Liberibacter solanacearum’ ( CL so). This study describes novel generic liberibacter genus primers, used to screen Australian psyllids for the presence of microflora that may confound diagnosis of exotic pathogens. CL br forms a unique clade in the liberibacter genus based on phylogenetic analysis of the 16S ribosomal ribonucleic acid ( rRNA ) region and multilocus sequence analysis ( MLSA ) of seven highly conserved genes, dnaG , gyrB , mutS , nusG , rplA , rpoB and tufB . The MLSA mapping approach described in this article was able to discriminate between two ‘ Ca . Liberibacter’ species within a metagenomic data set and represents a novel approach to detecting and differentiating unculturable species of liberibacter. Further, CL br can confound the Li et al . (2006) quantitative PCR ( qPCR ) diagnostic tests for CL as and CL af.

Summary ‘ Candidatus Accumulibacter’ is a biotechnologically important bacterial group that can accumulate large amounts of intracellular polyphosphate, contributing to biological phosphorus removal in wastewater treatment. Since its first molecular identification more than a decade ago, this bacterial group has drawn significant research attention due to its high abundance in many biological phosphorus removal systems. In the past 6 years, our understanding of Accumulibacter microbiology and ecophysiology has advanced rapidly, largely owing to genomic information obtained through shotgun metagenomic sequencing efforts. In this review, we focus on the metabolism, physiology, fine‐scale population structure and ecological distribution of Accumulibacter, aiming to integrate the information learned so far and to present a more complete picture of the microbiology of this important bacterial group.