The anaerobic fungi: challenges and opportunities for industrial lignocellulosic biofuel production

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation

The diving behaviour of mammal-eating killer whales (Orcinus orca): variations with ecological not physiological factors

Mammal-eating killer whales (Orcinus orca (L., 1758)) are a rare example of social predators that hunt together in groups of sexually dimorphic adults and juveniles with diverse physiological diving capacities. Day–night ecological differences should also affect diving as their prey show diel variation in activity and mammal-eating killer whales do not rely on echolocation for prey detection. Our objective was to explore the extent to which physiological aerobic capacities versus ecological factors shape the diving behaviour of this breath-hold diver. We used suction-cup-attached depth recorders (Dtags) to record 7608 dives of 11 animals in southeast Alaska. Analysis of dive sequences revealed a strong bout structure in both dive depth and duration. Day–night comparisons revealed reduced rates of deep dives, longer shallow dives, and shallower long-duration dives at night. In contrast, dive variables did not differ by age–sex class. Estimates of the aerobic dive limit (cADL) suggest that juveniles exceeded their cADL during as much as 15% of long dives, whereas adult males and females never exceeded their cADL. Mammal-eating killer whales in this area appear to employ a strategy of physiological compromise, with smaller group members diving nearer their physiological limits and large-bodied males scaling down their physiological performance

Ecological Invasion, Roughened Fronts, and a Competitor's Extreme Advance: Integrating Stochastic Spatial-Growth Models

Both community ecology and conservation biology seek further understanding of factors governing the advance of an invasive species. We model biological invasion as an individual-based, stochastic process on a two-dimensional landscape. An ecologically superior invader and a resident species compete for space preemptively. Our general model includes the basic contact process and a variant of the Eden model as special cases. We employ the concept of a "roughened" front to quantify effects of discreteness and stochasticity on invasion; we emphasize the probability distribution of the front-runner's relative position. That is, we analyze the location of the most advanced invader as the extreme deviation about the front's mean position. We find that a class of models with different assumptions about neighborhood interactions exhibit universal characteristics. That is, key features of the invasion dynamics span a class of models, independently of locally detailed demographic rules. Our results integrate theories of invasive spatial growth and generate novel hypotheses linking habitat or landscape size (length of the invading front) to invasion velocity, and to the relative position of the most advanced invader.Comment: The original publication is available at www.springerlink.com/content/8528v8563r7u2742

Dupilumab provides favourable long‐term safety and efficacy in children aged ≥ 6 to < 12 years with uncontrolled, severe atopic dermatitis: results from an open‐label phase IIa study and subsequent phase III open‐label extension study

Background Children aged ≥ 6 to < 12 years with severe atopic dermatitis (AD) have limited treatment options. In a 16‐week, randomized, placebo‐controlled, phase III trial in children, dupilumab, a monoclonal antibody inhibiting interleukin (IL)‐4/IL‐13 signalling, significantly improved signs and symptoms with acceptable safety; longer‐term safety and efficacy data are lacking. Objectives To report the pharmacokinetic profile and long‐term safety and efficacy of dupilumab in children (aged ≥ 6 to < 12 years) with severe AD. Methods Children (aged ≥ 6 to < 12 years) with severe AD were enrolled in a global, multicentre, phase IIa, open‐label, ascending‐dose, sequential cohort study and subsequent open‐label extension (OLE) study. Patients received single‐dose dupilumab 2 or 4 mg kg−1 followed by 8‐week pharmacokinetic sampling, then 2 or 4 mg kg−1 weekly for 4 weeks (phase IIa), followed by the same weekly regimen (OLE). Primary endpoints were dupilumab concentration–time profile and treatment‐emergent adverse events (TEAEs); secondary assessments included Eczema Area and Severity Index (EASI) and Peak Pruritus Numeric Rating Scale (PP‐NRS) score. Results Of 38 children enrolled, 37 completed phase IIa and 33 continued to the OLE. Nonlinear, target‐mediated pharmacokinetics characterized dupilumab concentrations (week 24–48 mean serum concentrations: 2 mg kg−1, 61–77 mg L−1; 4 mg kg−1, 143–181 mg L−1). TEAEs were mostly mild to moderate and transient; none led to treatment discontinuation. The most commonly reported TEAEs were nasopharyngitis (2 mg kg−1, 47%; 4 mg kg−1, 56%) and AD exacerbation (29% and 13%, respectively). Single‐dose dupilumab rapidly improved AD with further improvements through week 52. Mean EASI and PP‐NRS improved by −37%/−33% and −17%/−20% at week 2 (phase IIa) and −92%/−84% and −70%/−58% at week 52 (OLE), respectively. Conclusions These safety and efficacy results support the use of dupilumab as a continuous long‐term treatment for children aged ≥ 6 to < 12 years with severe AD

Top-Down enrichment guides in formation of synthetic microbial consortia for biomass degradation

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 cocultures 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 versus synthetic consortia underscore the difficulties in mimicking naturally occurring syntrophy in synthetic systems

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

Genomic and functional analyses of fungal and bacterial consortia that enable lignocellulose breakdown in goat gut microbiomes

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

Long-term efficacy and safety of dupilumab in adolescents with moderate-to-severe atopic dermatitis : results through week 52 from a phase III open-label extension trial (LIBERTY AD PED-OLE)

Background For adolescent patients (aged ≥ 12 to < 18 years) with uncontrolled moderate-to-severe atopic dermatitis (AD), 16 weeks of treatment with dupilumab resulted in substantial clinical benefit compared with placebo, with an acceptable safety profile. However, long-term data on the approved dose regimens of dupilumab in adolescents with AD are lacking. Objectives This open-label extension study (LIBERTY AD PED-OLE, NCT02612454) reports the long-term safety, efficacy, and pharmacokinetics of dupilumab in adolescents with moderate-to-severe AD who had participated in dupilumab parent trials. Methods Patients enrolled under the original study protocol received subcutaneous dupilumab according to a weight-based regimen (2 or 4 mg/kg every week). Following protocol amendment, patients were switched to subcutaneous dupilumab 300 mg every 4 weeks (q4w) irrespective of weight, and newly enrolled patients were started on dupilumab 300 mg q4w. Patients with an inadequate clinical response (Investigator’s Global Assessment [IGA] score of 0/1 was not reached) to the q4w regimen could be uptitrated to the approved dupilumab dose regimens of 200 or 300 mg every 2 weeks (body weight < 60 or ≥ 60 kg, respectively). Patients whose IGA score of 0/1 was maintained continuously for a 12-week period after week 40 were discontinued from dupilumab, monitored for relapse, and re-initiated on dupilumab if required. Results Data for 294 patients (mean age 14.7 years) were analyzed, 102 (34.7%) of whom had completed the 52-week visit at the database lock. The dupilumab long-term safety profile was comparable to that seen in adults and consistent with the known safety profile. Most treatment-emergent adverse events were mild/moderate. By week 52, 42.7% of patients had an IGA score of 0/1 (clear/almost clear), and 93.1%, 81.2%, and 56.4%, respectively, had at least a 50%, 75%, or 90% improvement in Eczema Area and Severity Index (EASI). Most (70.9%) patients required uptitration to the approved dupilumab dose regimen. The proportions of uptitrated patients with an IGA score of 0/1 or 75% improvement in EASI increased over time, reaching 35.7% and 51.9%, respectively, 48 weeks after the first uptitration visit. By week 52, 29.4% of patients had clear/almost clear skin sustained for 12 weeks and had stopped medication; 56.7% relapsed and were subsequently re-initiated on treatment, with a mean time to re-initiation of 17.5 (± standard deviation 17.3) weeks. Conclusions Consistent with results seen with short-term treatment, long-term treatment with dupilumab showed an acceptable safety profile while providing incremental clinical benefit with continued treatment over time. The high proportion of patients who needed uptitration because of inadequate response to q4w dosing supports the q2w dose regimen as optimal for this age group. Finally, the majority of patients who stopped medication after having clear/almost clear skin sustained over 12 weeks experienced disease recurrence, suggesting the need for continued dupilumab dosing to maintain efficacy. Trial Registration ClinicalTrials.gov Identifiers: NCT02612454, NCT02407756, NCT03054428, and NCT03050151

Experimentally validated reconstruction and analysis of a genome-scale metabolic model of an anaerobic Neocallimastigomycota fungus

Anaerobic gut fungi in the phylum Neocallimastigomycota typically inhabit the digestive tracts of large mammalian herbivores, where they play an integral role in the decomposition of raw lignocellulose into its constitutive sugar monomers. However, quantitative tools to study their physiology are lacking, partially due to their complex and unresolved metabolism that includes the largely uncharacterized fungal hydrogenosome. Modern omics approaches combined with metabolic modeling can be used to establish an understanding of gut fungal metabolism and develop targeted engineering strategies to harness their degradation capabilities for lignocellulosic bioprocessing. Here, we introduce a high-quality genome of the anaerobic fungus Neocallimastix lanati from which we constructed the first genome-scale metabolic model of an anaerobic fungus. Relative to its size (200 Mbp, sequenced at 62× depth), it is the least fragmented publicly available gut fungal genome to date. Of the 1,788 lignocellulolytic enzymes annotated in the genome, 585 are associated with the fungal cellulosome, underscoring the powerful lignocellulolytic potential of N. lanati. The genome-scale metabolic model captures the primary metabolism of N. lanati and accurately predicts experimentally validated substrate utilization requirements. Additionally, metabolic flux predictions are verified by 13C metabolic flux analysis, demonstrating that the model faithfully describes the underlying fungal metabolism. Furthermore, the model clarifies key aspects of the hydrogenosomal metabolism and can be used as a platform to quantitatively study these biotechnologically important yet poorly understood early-branching fungi