Editorial for Special Issue "Unleashing the Hidden Potential of Anaerobic Fungi".

Anaerobic fungi (AF) of the phylum Neocallimastigomycota are a very peculiar group of microorganisms [...].

No time to die: Comparative study on preservation protocols for anaerobic fungi.

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85-100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable.

Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats.

Anaerobic fungi (AF), belonging to the phylum Neocallimastigomycota, are a pivotal component of the digestive tract microbiome of various herbivorous animals. In the last decade, the diversity of AF has rapidly expanded due to the exploration of numerous (novel) habitats. Studies aiming at understanding the role of AF require robust and reliable isolation and cultivation techniques, many of which remained unchanged for decades. Using amplicon sequencing, we compared three different media: medium with rumen fluid (RF), depleted rumen fluid (DRF), and no rumen fluid (NRF) to enrich the AF from the feces of yak, as a rumen control; and Przewalski's horse, llama, guanaco, and elephant, as a non-rumen habitats. The results revealed the selective enrichment of Piromyces and Neocallimastix from the feces of elephant and llama, respectively, in the RF medium. Similarly, the enrichment culture in DRF medium explicitly manifested Piromyces-related sequences from elephant feces. Five new clades (MM1-5) were defined from llama, guanaco, yak, and elephant feces that could as well be enriched from llama and elephant samples using non-conventional DRF and NRF media. This study presents evidence for the selective enrichment of certain genera in medium with RF and DRF from rumen as well as from non-rumen samples. NRF medium is suggested for the isolation of AF from non-rumen environments.

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

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.

Why eDNA fractions need consideration in biomonitoring.

The analysis of environmental DNA (eDNA) is revolutionizing the monitoring of biodiversity as it allows to assess organismic diversity at large scale and unprecedented taxonomic detail. However, eDNA consists of an extracellular and intracellular fraction, each characterized by particular properties that determine the retrievable information on when and where organisms live or have been living. Here, we review the fractions of eDNA, describe how to obtain them from environmental samples and present a four-scenario concept that aims at enhancing spatial and temporal resolution of eDNA-based monitoring. Importantly, we highlight how the appropriate choice of eDNA fractions precludes misinterpretation of eDNA-based biodiversity data. Finally, future avenues of research towards eDNA fraction-specific analyses are outlined to unravel the full potential of eDNA-based studies targeting micro- and macro-organisms.

Microbial response on the first full-scale DEMON® biomass transfer for mainstream deammonification.

Sidestream partial nitritation and deammonification (pN/A) of high-strength ammonia wastewater is a well-established technology. Its expansion to the mainstream is, however mainly impeded by poor retention of anaerobic ammonia oxidizing bacteria (AnAOB), insufficient repression of nitrite oxidizing bacteria (NOB) and difficult control of soluble chemical oxygen demand and nitrite levels. At the municipal wastewater treatment plant in Strass (Austria) the microbial consortium was exhaustively monitored at full-scale over one and a half year with regular transfer of sidestream DEMON® biomass and further retention and enrichment of granular anammox biomass via hydrocyclone operation. Routine process parameters were surveyed and the response and evolution of the microbiota was followed by molecular tools, ex-situ activity tests and further, AnAOB quantification through particle tracking and heme measurement. After eight months of operation, the first anaerobic, simultaneous depletion of ammonia and nitrite was observed ex-situ, together with a direction to higher nitrite generation (68% of total NOx-N) as compared to nitrate under aerobic conditions. Our dissolved oxygen (DO) scheme allowed for transient anoxic conditions and had a strong influence on nitrite levels and the NOB community, where Nitrobacter eventually dominated Nitrospira. The establishment of a minor but stable AnAOB biomass was accompanied by the rise of Chloroflexi and distinct emergence of Chlorobi, a trend not seen in the sidestream system. Interestingly, the most pronounced switch in the microbial community and noticeable NOB repression occurred during unfavorable conditions, i.e. the cold winter season and high organic load. Further abatement of NOB was achieved through bioaugmentation of aerobic ammonia oxidizing bacteria (AerAOB) from the sidestream-DEMON® tank. Performance of the sidestream pN/A was not impaired by this operational scheme and the average volumetric nitrogen removal rate of the mainstream even doubled in the second half of the monitoring campaign. We conclude that a combination of both, regular sidestream-DEMON® biomass transfer and granular SRT increase via hydrocyclone operation was crucial for AnAOB establishment within the mainstream.

The masking effect of extracellular DNA and robustness of intracellular DNA in anaerobic digester NGS studies: A discriminatory study of the total DNA pool.

Most commonly, next generation sequencing-based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular- (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA-derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

CoMA - an intuitive and user-friendly pipeline for amplicon-sequencing data analysis.

In recent years, there has been a veritable boost in next-generation sequencing (NGS) of gene amplicons in biological and medical studies. Huge amounts of data are produced and need to be analyzed adequately. Various online and offline analysis tools are available; however, most of them require extensive expertise in computer science or bioinformatics, and often a Linux-based operating system. Here, we introduce "CoMA-Comparative Microbiome Analysis" as a free and intuitive analysis pipeline for amplicon-sequencing data, compatible with any common operating system. Moreover, the tool offers various useful services including data pre-processing, quality checking, clustering to operational taxonomic units (OTUs), taxonomic assignment, data post-processing, data visualization, and statistical appraisal. The workflow results in highly esthetic and publication-ready graphics, as well as output files in standardized formats (e.g. tab-delimited OTU-table, BIOM, NEWICK tree) that can be used for more sophisticated analyses. The CoMA output was validated by a benchmark test, using three mock communities with different sample characteristics (primer set, amplicon length, diversity). The performance was compared with that of Mothur, QIIME and QIIME2-DADA2, popular packages for NGS data analysis. Furthermore, the functionality of CoMA is demonstrated on a practical example, investigating microbial communities from three different soils (grassland, forest, swamp). All tools performed well in the benchmark test and were able to reveal the majority of all genera in the mock communities. Also for the soil samples, the results of CoMA were congruent to those of the other pipelines, in particular when looking at the key microbial players.

Quantities of Intra- and Extracellular DNA Reveal Information About Activity and Physiological State of Methanogenic Archaea.

Although being a common aim of many microbial ecology studies, measuring individual physiological conditions of a microbial group or species within a complex consortium is still a challenge. Here, we propose a novel approach that is based on the quantification of sequentially extracted extracellular (exDNA) and intracellular DNA (iDNA) and reveals information about cell lysis and activity of methanogenic archaea within a biogas-producing microbial community. We monitored the methane production rates of differently treated batch anaerobic cultures and compared the concentrations of the alpha subunit of the methyl coenzyme M reductase gene of methanogenic archaea in extracellular and intracellular DNA fractions and in the classically extracted total DNA pool. Our results showed that this fine-tuned DNA approach coupled with the interpretation of the ratio between free exDNA and iDNA considerably improved microbial activity tracking compared to the classical extraction/quantification of total DNA. Additionally, it allowed to identify and quantify methanogenic populations that are inactive and those that are strongly influenced by cell lysis. We argue that despite the need of further studies, this method represents a novel approach to gain specific physiological information from a complex environmental sample and holds the potential to be applied to other microbes of interest.

Employing anaerobic fungi in biogas production: challenges & opportunities.

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to efficiently break down lignocellulosic biomass. Their unique combination of mechanical and enzymatic attacks on recalcitrant plant structures bears great potential for enhancement of the anaerobic digestion (AD) process. Although scientists in this field have long agreed upon the potential of AF for biotechnology, research is only recently gaining traction. This delay was largely due to difficulties in culture-dependent and culture-independent analysis of those high-maintenance organisms with their still unknown complex growth requirements. In this review, we will summarize current research efforts on bioaugmentation with AF and further point out, how the lack of basic knowledge on AF nutritional needs hampers their implementation on an industrial scale. Through this, we hope to further kindle interest into basic research on AF in order to advance their stable integration into biotechnological processes.

Biomethanation at 45 °C offers high process efficiency and supports hygienisation.

The aim of this work was to prove a process temperature of 45 °C as a practical alternative to commonly applied mesophilic (37 °C) and thermophilic (55 °C) anaerobic digestion (AD). Regarding methane production, no differences were found between the three temperature regimes. However, the maximum possible loading rate at 45 °C exceeded that at 37 °C and 55 °C. Pathogen inactivation at 45 °C was higher than at 37 °C and similarly efficient as at 55 °C. At each process temperature, a unique microbial community established. In addition, the archaeome at 55 °C was dominated by hydrogenotrophs, while at 37 °C and 45 °C it was dominated by acetotrophs. For the investigated substrate mixture, liquid cattle manure with wheat straw as co-substrate, 45 °C turned out to be preferable for AD. For other substrates, these findings still need to be confirmed.

Simple yet effective: Microbial and biotechnological benefits of rumen liquid addition to lignocellulose-degrading biogas plants.

In biogas plants, lignocellulose-rich biomass (LCB) is particularly slowly degraded, causing high hydraulic retention times. This fact lowers the interests for such substrates. To enhance LCB-degradation, cattle rumen fluid, a highly active microbial resource accruing in the growing meat industry, might be used as a potential source for bioaugmentation. This study compares 0%, 20% and 40% rumen liquid in a batch anaerobic digestion approach. Moreover, it determines the biogas- and methane-potentials as well as degradation-speeds of corn straw, co-digested with cattle manure. It inspects microbial communities via marker-gene sequencing, qPCR and RNA-DGGE and draws attention on possible beneficial effects of rumen addition on the biogas-producing community. Bioaugmentation with 20% and 40% v/v rumen liquid accelerated methane yields by 5 and 6 days, respectively (i.e. reaching 90% of total methane production). It also enhanced LCB- as well as (hemi)cellulose- and volatile fatty acid degradation. These results are supported by increased abundances of bacteria, methanogens and anaerobic fungi in treatments with rumen liquid amendment, and point towards the persistence of specific rumen-borne microorganisms especially during the first phase of the experiment. The results suggest that rumen liquid addition is a promising strategy for enhanced and accelerated exploitation of LCB for biomethanisation.

Temperature shapes the microbiota in anaerobic digestion and drives efficiency to a maximum at 45 °C.

Throwing longstanding habits over the pile may be necessary to improve biogas production, in particular when it comes to the process temperature. Its effect on biogas production was investigated with lab-scale reactors operated in fed-batch mode (cattle slurry and maize straw) at 10-55 °C over six months. Biochemical and microbial changes were comprehensively investigated. Production was highest and most efficient at 45 °C with an average methane yield of 166 NL kg-1 VS, and thus 12.8% and 9.6% higher than at 37 and 55 °C. Temperature significantly affected the microbiota and higher temperature provoked a shift from Bacteroidetes/Proteobacteria to Firmicutes. A transition from hydrogenotrophic to acetoclastic methanogenesis was observed from 10 to 45 °C, while the trend was reversed at 55 °C. The results contest the textbook notion of preferred and most efficient temperatures for AD and suggest reconsideration of the temperature range around 45 °C for efficient manure-based co-fermentation.

The use of extracellular DNA as a proxy for specific microbial activity.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

Anaerobic Fungi and Their Potential for Biogas Production.

Plant biomass is the largest reservoir of environmentally friendly renewable energy on earth. However, the complex and recalcitrant structure of these lignocellulose-rich substrates is a severe limitation for biogas production. Microbial pro-ventricular anaerobic digestion of ruminants can serve as a model for improvement of converting lignocellulosic biomass into energy. Anaerobic fungi are key players in the digestive system of various animals, they produce a plethora of plant carbohydrate hydrolysing enzymes. Combined with the invasive growth of their rhizoid system their contribution to cell wall polysaccharide decomposition may greatly exceed that of bacteria. The cellulolytic arsenal of anaerobic fungi consists of both secreted enzymes, as well as extracellular multi-enzyme complexes called cellulosomes. These complexes are extremely active, can degrade both amorphous and crystalline cellulose and are probably the main reason of cellulolytic efficiency of anaerobic fungi. The synergistic use of mechanical and enzymatic degradation makes anaerobic fungi promising candidates to improve biogas production from recalcitrant biomass. This chapter presents an overview about their biology and their potential for implementation in the biogas process.

A novel fixed fibre biofilm membrane process for on-site greywater reclamation requiring no fouling control.

On-site greywater treatment and reuse in urban areas bears the potential to reduce huge quantities of wastewater and lower freshwater shortages. Until now dissemination of small, single household applications has been rather limited as simple and high quality water producing, but also cost-effective treatment units have not been developed so far. This paper proposes a new process, based on a concurrently working hollow-fibre membrane as fixed biofilm support and filtration device. Bioreactor characteristics, influence of different aeration rates, membrane flux development, as well as structure and composition of biofilm were monitored to evaluate the performance of the tested pilot unit. The introduced process achieved international water reuse guidelines, worked soundly and could, compared to conventional micro MBR, significantly reduce energy demand (<1.4 kWh m(-3)). Fouling control by air scouring and chemical cleaning was not required once flux had stabilized. The biofilm analysis showed a porous, spongy-like structure. Microbiological investigation revealed a community of sheathed bacteria and nematodes that could play an important role in the flux stabilisation effect. In general, the study confirmed the suitability of the presented process for greywater treatment and provides valuable design data for future optimization and systematic analysis.

Finding a robust strain for biomethanation: anaerobic fungi (Neocallimastigomycota) from the Alpine ibex (Capra ibex) and their associated methanogens.

Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.