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Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates. 

Development of the bioeconomy is driven by our ability to access the energy‐rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value‐added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two‐stage bioprocessing system pairing the anaerobic fungusPiromyces indianaewith the yeastKluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose.P.indianaeefficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, whichK.marxianusconsumed while producing 2.4 g/L ethyl acetate. An engineered strain ofK.marxianuswas also able to produce 550 mg/L 2‐phenylethanol and 150 mg/L isoamyl alcohol fromP.indianaehydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

 

Anaerobic fungi are among the most active plant‐degrading microbes in nature. Increased insight into the mechanisms and environmental cues that regulate fungal hydrolysis would better inform bioprocessing strategies to depolymerize lignocellulose. Here, we compare the response of three strains of anaerobic fungi (Piromyces finnis,Anaeromyces robustus, andNeocallimastix californiae) to catabolite regulation by simple carbohydrates. Anaerobic fungi exhibited high enzymatic activity against crystalline cellulose, which was repressed upon incubation with free sugars. Cellulolytic degradation was also inhibited when fungi were exposed to sugars they did not metabolize, suggesting a general mode of catabolite repression. RNA‐Seq experiments in the presence of excess glucose confirmed repression of carbohydrate active enzymes during sugar uptake, and offer a path towards unmasking the function of co‐regulated genes that could be involved in biomass degradation. Overall, these results suggest that sugar‐rich hydrolysates tune the behavior of anaerobic fungi by dampening production of their biomass‐degrading enzymes. © 2018 American Institute of Chemical EngineersAIChE J, 64: 4263–4270, 2018

 

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 ofSaccharomyces 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.