Marine macroalgae is a biomass resource for the manufacture of fuels and chemicals, which can be sustainably harvested from seaweed farms or from man-made structures where it accumulates as a biofouling organism. However, in temperate regions farmed macroalgae can only be harvested between late Spring and early Summer, limiting year-round availability. Here we show that a conventional grass ensilage procedure preserves Saccharina latissima dominated biomass on the tonne scale for 30 months, enabling year-round use of this biomass. Following processing, the resulting dried and pelletised ensiled macroalgae material was subject to ThermoCatalytic ReformingTM, comprising sequential pyrolysis (450 degrees C) and either dry or steam catalytic reforming (700 degrees C) processes. Both processing methods produced a mixture of bio-oil (1.6-1.9 wt%) and hydrogen-rich permanent gases (30.9-31.1 wt%) with higher heating values of 34.8-35.4 MJ/kg and 18.0-24.2 MJ/m3, respectively, together with char (45.5-48.5 % wt). The permanent gases can be used directly for heat generation, while hydro-treatment of the bio-oil would afford a material that can be blended with traditional transport fuels. This work demonstrates that if operated at scale, the combined harvesting, ensilaging and Thermo-Catalytic ReformingTM of preserved macroalgal biomass offers a year-round decentralised energy resource.

Two protocols involving batch cultures were used to investigate the bioaugmentation of methane production by Pecoramyces ruminantium, and Methanobrevibacter thaueri. Protocol I examined the effect of altering the proportion of the microbial constituents in inoculum on alfalfa stalk fermentations and showed a 25 % improvement in dry matter loss in cultures where the inoculum contained just 30 % of co-culture and 70 % of fungal monoculture. Protocol II involved consecutive cultures and alternating inoculations. This protocol resulted in 17-22 mL/g DM methane production with co-cultures a 30 % increase in methane relative to the fungal monoculture. Both protocols indicate that the co-culture rapidly dominated and was more resilient than the monoculture. Synergistic interaction between fungus and methanogen, promoted more efficient lignocellulose degradation and higher methane yield. This study highlighted the potential of microbial co-cultures for enhancing methane production from lignocellulosic biomass, offering a promising bioaugmentation strategy for improving biogas yields and waste valorization.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

Recent genomic analyses have revealed that anaerobic gut fungi possess both the largest number and highest diversity of lignocellulolytic enzymes of all sequenced fungi, explaining their ability to decompose lignocellulosic substrates, e.g., agricultural waste, into fermentable sugars. Despite their potential, the development of engineering methods for these organisms has been slow due to their complex life cycle, understudied metabolism, and challenging anaerobic culture requirements.

Digestates from anaerobic digestion (AD) of food waste contain fertiliser nutrients (such as P and N) which are valuable for agricultural purposes and can be environmentally hazardous if disposal is uncontrolled. Here, we applied electrocoagulation (EC) for treatment of digestates, to separate liquids and nutrient-rich solids. Coagulant-dosing electrocoagulation (CDEC) was used to compare Al and steel anodes for treatment of digestate from AD fermenters fed a controlled diet representative of food waste. When applying metal dosing concentrations of 0–4.66 mM, Fe was found to be up to 29.8 % superior to Al in terms of aiding removal of chemical oxygen demand (COD). To mitigate plate fouling, the digestate was diluted to 0.25 and 0.1 concentrations to enable successful treatment by continuous-flow electrocoagulation (CFEC). The highest recovery of soluble P per Fe added by CFEC was 22.4 mg-P g-Fe−1. This was achieved when using 2.33 mM Fe to treat a 0.25 dilution of digestate. In comparison to a control, these optimal conditions also caused a reduction of 4.5 mg L−1 (or 87.7 %) of soluble P and the removal of 254 mg L−1 (33.2 %) of COD in the filtrate. The NH4+ concentration in filtrate was not influenced by EC treatment. Analyses of a range of known toxic elements (Cu, Ni, Zn, As, Cd, Cr, Mo and Pb) indicates that the solids recovered by CFEC could be suitable for application to land.

The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment experiments from goat faeces to determine how substrate and antibiotic selection influence membership, activity, stability and chemical productivity of herbivore gut communities. We assembled 719 high-quality metagenome-assembled genomes (MAGs) that are unique at the species level. More than 90% of these MAGs are from previously unidentified herbivore gut microorganisms. Microbial consortia dominated by anaerobic fungi outperformed bacterially dominated consortia in terms of both methane production and extent of cellulose degradation, which indicates that fungi have an important role in methane release. Metabolic pathway reconstructions from MAGs of 737 bacteria, archaea and fungi suggest that cross-domain partnerships between fungi and methanogens enabled production of acetate, formate and methane, whereas bacterially dominated consortia mainly produced short-chain fatty acids, including propionate and butyrate. Analyses of carbohydrate-active enzyme domains present in each anaerobic consortium suggest that anaerobic bacteria and fungi employ mostly complementary hydrolytic strategies. The division of labour among herbivore anaerobes to degrade plant biomass could be harnessed for industrial bioprocessing.

Chicken manure is an agricultural by-product that is a problematic feedstock for anaerobic digestion due to its high nitrogen content inhibiting methane yields. This research examines a novel pilot-scale method of ammonia stripping, the nitrogen recovery process (NRP) developed by Alchemy Utilities Ltd. The NRP was designed to remove and recover nitrogen from chicken manure and two different operating conditions were examined. Both operating conditions demonstrated successful nitrogen removal and recovery. The biochemical methane potential assays were used to compare the digestibility of the NRP-treated chicken manures to that of a fresh chicken manure control. Overall, the biochemical methane potential assays demonstrated that some NRP-treated chicken manure treatments produced significantly more methane compared to untreated manure, with no inhibition occurring in relation to ammonium. However, some of the NRP-treated chicken manures produced similar or lower methane yields compared to fresh chicken manure. The NRP requires further development to improve the efficiency of the pilot-scale unit for commercial-scale operation and longer-term continuous anaerobic digestion trials are required to determine longer-term methane yield and ammonium inhibition effects. However, these initial results clearly demonstrate the technology’s potential and novel application for decentralised, on-farm nitrogen recovery and subsequent anaerobic digestion of chicken manure.

Oat husks are produced during the milling process of oats. Oat husks are a lignocellulosic material that have the potential for valorization thereby improving the circular economy of agricultural by-products. However, due to the high lignocellulosic content, there are limited valorization pathways for oat husks. To improve the anaerobic digestibility of oat husks, pre-treatment was investigated as a method to aid valorization. A novel extrusion process was used in an attempt to fragment the lignocellulosic structure of oat husks prior to anaerobic digestion. The extrusion pre-treatment was investigated to determine the effect it may have on altering the methane yield and digestibility of oat husks. Biochemical methane potential assays were undertaken using oat husks with no pre-treatment and extruded oat husks. These assays demonstrated that extruded oat husks produced a significantly higher methane yield of 264 ml/gVS fed, which was 27% greater than the methane yield produced from the untreated oat husks. Similarly, the total solids degradation was also significantly higher for extruded oat husks treatment compared to the untreated oat husks. Overall, the extrusion process demonstrated an increased methane yield for oat husks compared to previously published data. The biomethane potential tests suggest that extruded oat husks would be a feedstock suitable for anaerobic digestion.

Measuring the growth rate of non-model anaerobic microbes typically requires the use of time-consuming and often destructive manual measurements. Here, an Arduino based automatic pressure evaluation system (A-APES) was developed to automatically measure the rate of fermentation gas production as a proxy for microbial growth in anaerobic systems. The A-APES system measures accumulated gas pressure in sealed cultures accurately at high-resolution, while venting the system at programmed intervals to prevent over pressurization. The utility of A-APES is demonstrated in this study by quantifying the growth rate and phases of a biomass-degrading anaerobic gut fungus, which cannot be otherwise measured via conventional techniques due to its association with particulate substrates. Given the utility of the A-APES approach, we provide a complete construction guide to fabricate the device, which is three times less expensive compared to existing commercial alternatives.

Pyrolysis char residues from ensiled macroalgae were examined to determine their potential as growth promoters on germinating and transplanted seedlings. Macroalgae was harvested in May, July and August from beach collections, containing predominantly Laminaria digitata and Laminaria hyperborea; naturally seeded mussel lines dominated by Saccharina latissima; and lines seeded with cultivated L. digitata. Material was ensiled, pressed to pellets and underwent pyrolysis using a thermo-catalytic reforming (TCR) process, with and without additional steam. The chars generated were then assessed through proximate and ultimate analysis. Seasonal changes had the prevalent impact on char composition, though using mixed beach-harvested material gave a greater variability in elements than when using the offshore collections. Applying the char at 5% (v/v)/2% (w/w) into germination or seedling soils was universally negative for the plants, inhibiting or delaying all parameters assessed with no clear advantage in harvesting date, species or TCR processing methodology. In germinating lettuce seeds, soil containing the pyrolysis chars caused a longer germination time, poorer germination, fewer true leaves to be produced, a lower average plant health score and a lower final biomass yield. For transplanted ryegrass seedlings, there were lower plant survival rates, with surviving plants producing fewer leaves and tillers, lower biomass yields when cut and less regrowth after cutting. As water from the char-contained plant pots inhibited the lettuce char control, one further observation was that run-off water from the pyrolysis char released compounds which detrimentally affected cultivated plant growth. This study clearly shows that pyrolysed macroalgae char does not fit the standard assumption that chars can be used as soil amendments at 2% (w/w) addition levels. As the bioeconomy expands in the future, the end use of residues and wastes from bioprocessing will become a genuine global issue, requiring consideration and demonstration rather than hypothesized use.

Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host’s ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results.

The dairy processing and slaughterhouse industries consume large volumes of water and their waste effluents often contain high levels of chemical oxygen demand (COD), biochemical/biological oxygen demand (BOD) and fertilising nutrients. Therefore, water treatment is necessary to reduce the levels of these contaminants prior to discharge or reuse of the water. In this short review paper we provide a brief overview of electrocoagulation (EC) technology and summarise the current literature relating to the use of EC treatment to clean dairy processing and slaughterhouse wastewaters.

Consortium-based approaches are a promising avenue toward efficient bioprocessing. However, many complex microbial interactions dictate community dynamics and stability that must be replicated in synthetic systems. The rumen and/or hindguts of large mammalian herbivores harbor complex communities of biomass-degrading fungi and bacteria, as well as archaea and protozoa that work collectively to degrade lignocellulose, yet the microbial interactions responsible for stability, resilience and activity of the community remain largely uncharacterized. In this work, we demonstrate a "top-down" enrichment-based methodology for selecting a minimal but effective lignocellulose-degrading community that produces methane-rich fermentation gas (biogas). The resulting enrichment consortium produced 0.75-1.9-fold more fermentation gas at 1.4-2.1 times the rate compared to a monoculture of fungi from the enrichment. Metagenomic sequencing of the top-down enriched consortium revealed genomes encoding for functional compartmentalization of the community, spread across an anaerobic fungus (Piromyces), a bacterium (Sphaerochaeta), and two methanogenic archaea (Methanosphaera and Methanocorpusculum). Guided by the composition of the top-down enrichment, several synthetic co-cultures were formed from the "bottom-up" using previously isolated fungi, Neocallimastix californiae and Anaeromyces robustus paired with the methanogen Methanobacterium bryantii. While cross-feeding occurred in synthetic co-cultures, removal of fungal metabolites by methanogens did not increase the rate of gas production or the rate of substrate deconstruction by the synthetic community relative to fungal monocultures. Metabolomic characterization verified that syntrophy was established within synthetic co-cultures, which generated methane at similar concentrations compared to the enriched consortium but lacked the temporal stability (resilience) seen in the native system. Taken together, deciphering the membership and metabolic potential of an enriched gut consortium enables the design of methanogenic synthetic co-cultures. However, differences in the growth rate and stability of enriched vs. synthetic consortia underscore the difficulties in mimicking naturally occurring syntrophy in synthetic systems.

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 degrees 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 Nm(3) kg-VS-1 versus a range of 0.14-0.58 Nm(3) kg-VS-1 for other treatments), with the highest percentage of methane in total biogas (61.6% versus a range of 36.155.2% 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 log(10) CFU g-TS-1), whilst Clostridium perfringens levels remained unaffected by treatment throughout the digestion process (5.3 +/- 0.2 log(10) CFU g-TS-1). (C) 2018 Elsevier Ltd. All rights reserved.

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.

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.

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.