Michael K. Theodorou

Temporal Metagenomic and Metabolomic Characterization of Fresh Perennial Ryegrass Degradation by Rumen Bacteria

Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within “metabolism;” predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers.

Dewatering treatments to increase dry matter content of the brown seaweed, kelp (Laminaria digitata ((Hudson) JV Lamouroux)).

Macroalgal water content is an on-going problem for the use of readily accessible seaweeds in sustainable biorefining, including fuel production. Silage is a reduced-water, compactable, easily stored, transportable material. Ensiling could establish a non-seasonal supply of preserved algal biomass, but requires high initial dry matter content to mitigate environmental pollution risks from effluent. This study investigated potential dewatering methods for kelp harvested throughout the year. Treatments included air-drying, osmotic media and acids. Significant interactions between treatment and harvest-time were observed for traits of interest. Fresh weight loss during treatment was composed of changes in water and dry matter content. Air-drying gave reliable increase in final dry matter content; in summer and autumn 30% dry matter content was reached after 24h. Dilute hydrochloric acid reduced stickiness and rendered material suitable for dewatering by screw-pressing; it may be possible to use the consequent pH reduction to promote efficient preservation.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

The conversion of lignocellulose‐rich biomass to bio‐based chemicals and higher order fuels remains a grand challenge, as single‐microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar‐rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value‐added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.

Transcriptomic characterization of Caecomyces churrovis : a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus

Anaerobic gut fungi are the primary colonizers of plant material in the rumen microbiome, but are poorly studied due to a lack of characterized isolates. While most genera of gut fungi form extensive rhizoidal networks, which likely participate in mechanical disruption of plant cell walls, fungi within the Caecomyces genus do not possess these rhizoids. Here, we describe a novel fungal isolate, Caecomyces churrovis, which forms spherical sporangia with a limited rhizoidal network yet secretes a diverse set of carbohydrate active enzymes (CAZymes) for plant cell wall hydrolysis. Despite lacking an extensive rhizoidal system, C. churrovis is capable of growth on fibrous substrates like switchgrass, reed canary grass, and corn stover, although faster growth is observed on soluble sugars. Gut fungi have been shown to use enzyme complexes (fungal cellulosomes) in which CAZymes bind to non-catalytic scaffoldins to improve biomass degradation efficiency. However, transcriptomic analysis and enzyme activity assays reveal that C. churrovis relies more on free enzymes compared to other gut fungal isolates. Only 15% of CAZyme transcripts contain non-catalytic dockerin domains in C. churrovis, compared to 30% in rhizoid-forming fungi. Furthermore, C. churrovis is enriched in GH43 enzymes that provide complementary hemicellulose degrading activities, suggesting that a wider variety of these activities are required to degrade plant biomass in the absence of an extensive fungal rhizoid network. Overall, molecular characterization of a non-rhizoid-forming anaerobic fungus fills a gap in understanding the roles of CAZyme abundance and associated degradation mechanisms during lignocellulose breakdown within the rumen microbiome.

The anaerobic digestion of pig carcase with or without sugar beet pulp, as a novel on-farm disposal method

Anaerobic digestion was investigated as a potential method for on-farm disposal of fallen stock (pig carcases), degrading the carcase material to produce biogas and digestate. The effects of feedstock (sugar beet pulp or pig carcase material or a 50:50 mix) and organic loading rate (50 g-TS L-1 or 100 g-TS L-1), during mesophilic (35 °C) anaerobic digestion were investigated. Anaerobic digestion was achieved for all experimental treatments, however the pig carcase material at the higher organic loading rate produced the second highest methane yield (0.56 Nm3 kg-VS-1 versus a range of 0.14-0.58 Nm3 kg-VS-1 for other treatments), with the highest percentage of methane in total biogas (61.6% versus a range of 36.1-55.2% for all other treatments). Satisfactory pathogen reduction is a legislative requirement for disposal of carcase material. Pathogens were quantified throughout the anaerobic digestion process. Enterococcus faecalis concentrations decreased to negligible levels (2.8 log10 CFU g-TS-1), whilst Clostridium perfringens levels remained unaffected by treatment throughout the digestion process (5.3 ± 0.2 log10 CFU g-TS-1).

Methods for Genomic Characterization and Maintenance of Anaerobic Fungi

The rapid development of molecular biology and bioinformatics has fueled renewed interests in anaerobic fungi from the phylum Neocallimastigomycota. This chapter presents well-established methods for isolation, routine cultivation, and cryopreservation of anaerobic fungi. Moreover, detailed nucleic acid extraction protocols are provided, which should enable readers to isolate high-quality DNA and RNA from a variety of anaerobic fungal culture media for downstream applications such as next-generation sequencing.

Species variation in the effects of dewatering treatment on macroalgae

Seaweeds can be a valuable resource for biorefinery and biotechnology applications, but their high water content is a recurrent problem and one of the key bottlenecks for their sustainable use. Treatments to increase dry matter content of the kelp Laminaria digitata were recently described by the authors. However macroalgae are an extremely diverse group of organisms and compositional variation between species may influence the effects of particular treatments. In this study, potential dewatering treatments including drying, osmotic media, and the application of both organic and mineral acids all followed by screw-pressing have been tested on two other species of kelp (Laminaria hyperborea and Saccharina latissima) and a red seaweed (Palmaria palmata). Conditions that dewatered these species were identified and the data have been combined with the previous results for L. digitata. There were significant differences between species across all the traits of interest. However dewatering was highly dependent on specific interactions with both treatment and season of collection. Nevertheless, the dry matter content of brown seaweeds was widely and successfully increased by air drying or acid treatment followed by screw-pressing. The results for P. palmata were quite different, particularly with regard to juice production. For this species, acid treatment did not result in dewatering, but dry matter content could be increased by screw-pressing immediately after harvest. Together the data presented here demonstrate that dewatering pre-treatments need to be specific for the type of seaweed to be processed; important knowledge for the future use of this sustainable biomass resource.