Unleashing the Hidden Potential of Anaerobic Fungi

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Environmental Microbiology".

Deadline for manuscript submissions: closed (15 September 2021) | Viewed by 35434

Special Issue Editors

Microbial Resource Management, Institute of Microbiology, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria
Interests: anaerobic fungi; soil; biogas; environmental microbiology; microbial diversity; molecular microbial ecology; water quality
Institute of Chemistry and Biotechnology (ICBT), Zurich University of Applied Sciences (ZHAW), Zurich, Switzerland
Interests: biogas; biowaste valorisation; bioprocess technology
Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, 85354 Freising, Germany
Interests: molecular microbial ecology; applied anaerobic microbiology; phylogenetics; biogas processes; hygienization by biogas technology; renewable energies

Special Issue Information

Dear Colleagues,

Anaerobic fungi (AF) are a very peculiar group of microorganisms and the sole fungal phylum exhibiting an anaerobic lifestyle and some ancestral features lost in higher-evolved fungi. Since their first discovery in the early nineteen-hundreds and correct affiliation to the kingdom of fungi in 1975 by Orpin, many researchers have delved into these highly potent degraders of lignocellulosic biomass (LCB). Their panoply of hydrolytic enzymes makes them key players in the digestive tract of herbivores; however, their worldwide distribution seems not to be restricted to this habitat alone. Despite the plenitude of research on this group, many questions still remain unanswered, and implementation of AF to, e.g., biomethanation of LCB or bioethanol production is still in its infancy. This is where international projects like (https://www.hipoaf.com) hook in and aim at answering basic questions such as ideal growth conditions, improved and novel detection techniques, screening for novel habitats, strains and enzymes, symbiotic interactions of AF, and, eventually, at paving the way to successful biotechnological implementation of these unique microorganisms. These questions are also the scope of this Special Issue that is dedicated to updates on all fields related to AF research in agriculture, biotechnology and AF systematics, physiology, gene evolution, horizontal gene transfer, and molecular detection.

Dr. Sabine Marie Podmirseg
Dr. Michael Lebuhn
Prof. Dr. Urs Baier
Guest Editors

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Keywords

  • neocallimastigomycota
  • break-up of lignocellulosic biomass
  • novel habitats
  • cultivation of anaerobic fungi
  • diversity
  • growth requirements
  • synergistic interactions
  • host organisms and habitats
  • detection techniques
  • fungal gene evolution and HGT

Published Papers (11 papers)

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Editorial

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3 pages, 187 KiB  
Editorial
Editorial for Special Issue “Unleashing the Hidden Potential of Anaerobic Fungi”
Microorganisms 2023, 11(3), 652; https://doi.org/10.3390/microorganisms11030652 - 03 Mar 2023
Viewed by 909
Abstract
Anaerobic fungi (AF) of the phylum Neocallimastigomycota are a very peculiar group of microorganisms [...] Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)

Research

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15 pages, 1556 KiB  
Article
Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats
Microorganisms 2022, 10(10), 1972; https://doi.org/10.3390/microorganisms10101972 - 05 Oct 2022
Cited by 2 | Viewed by 1671
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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23 pages, 5724 KiB  
Article
Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages
Microorganisms 2022, 10(9), 1749; https://doi.org/10.3390/microorganisms10091749 - 30 Aug 2022
Cited by 7 | Viewed by 2446
Abstract
Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. [...] Read more.
Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara). Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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16 pages, 2918 KiB  
Article
Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices
Microorganisms 2021, 9(9), 1986; https://doi.org/10.3390/microorganisms9091986 - 18 Sep 2021
Cited by 3 | Viewed by 2155
Abstract
Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of [...] Read more.
Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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18 pages, 5698 KiB  
Article
Isolation and Biochemical Characterization of Six Anaerobic Fungal Strains from Zoo Animal Feces
Microorganisms 2021, 9(8), 1655; https://doi.org/10.3390/microorganisms9081655 - 03 Aug 2021
Cited by 10 | Viewed by 2631
Abstract
Anaerobic fungi are prime candidates for the conversion of agricultural waste products to biofuels. Despite the increasing interest in these organisms, their growth requirements and metabolism remain largely unknown. The isolation of five strains of anaerobic fungi and their identification as Neocallimastix cameroonii [...] Read more.
Anaerobic fungi are prime candidates for the conversion of agricultural waste products to biofuels. Despite the increasing interest in these organisms, their growth requirements and metabolism remain largely unknown. The isolation of five strains of anaerobic fungi and their identification as Neocallimastix cameroonii, Caecomyces spec., Orpinomyces joyonii, Pecoramyces ruminantium, and Khoyollomyces ramosus, is described. The phylogeny supports the reassignment of Neocallimastix californiae and Neocallimastix lanati to Neocallimastix cameroonii and points towards the redesignation of Cyllamyces as a species of Caecomyces. All isolated strains including strain A252, which was described previously as Aestipascuomyces dubliciliberans, were further grown on different carbon sources and the produced metabolites were analyzed; hydrogen, acetate, formate, lactate, and succinate were the main products. Orpinomyces joyonii was lacking succinate production and Khoyollomyces ramosus was not able to produce lactate under the studied conditions. The results further suggested a sequential production of metabolites with a preference for hydrogen, acetate, and formate. By comparing fungal growth on monosaccharides or on the straw, a higher hydrogen production was noticed on the latter. Possible reactions to elevated sugar concentrations by anaerobic fungi are discussed. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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21 pages, 2253 KiB  
Article
The Effect of a High-Grain Diet on the Rumen Microbiome of Goats with a Special Focus on Anaerobic Fungi
Microorganisms 2021, 9(1), 157; https://doi.org/10.3390/microorganisms9010157 - 12 Jan 2021
Cited by 15 | Viewed by 3506
Abstract
This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain; n = 4) for 20 days [...] Read more.
This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain; n = 4) for 20 days and were then abruptly shifted to a high-grain (HG) diet (40% corn grain, 60% AH; n = 4) and treated for another 10 days. Rumen content samples were collected from the cannulated animals at the end of each diet period (day 20 and 30). The microbiome structure was studied using high-throughput sequencing for bacteria, archaea (16S rRNA gene) and fungi (ITS2), accompanied by qPCR for each group. To further elucidate unclassified AF, clone library analyses were performed on the ITS1 spacer region. Rumen pH was significantly lower in HG diet fed goats, but did not induce subacute ruminal acidosis. HG diet altered prokaryotic communities, with a significant increase of Bacteroidetes and a decrease of Firmicutes. On the genus level Prevotella 1 was significantly boosted. Methanobrevibacter and Methanosphaera were the most abundant archaea regardless of the diet and HG induced a significant augmentation of unclassified Thermoplasmatales. For anaerobic fungi, HG triggered a considerable rise in Feramyces observed with both ITS markers, while a decline of Tahromyces was detected by ITS2 and decrease of Joblinomyces by ITS1 only. The uncultured BlackRhino group revealed by ITS1 and further elucidated in one sample by LSU analysis, formed a considerable part of the AF community of goats fed both diets. Results strongly indicate that the rumen ecosystem still acts as a source for novel microorganisms and unexplored microbial interactions and that initial rumen microbiota of the host animal considerably influences the reaction pattern upon diet change. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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15 pages, 2021 KiB  
Article
Cut-Lengths of Perennial Ryegrass Leaf-Blades Influences In Vitro Fermentation by the Anaerobic Fungus Neocallimastix frontalis
Microorganisms 2020, 8(11), 1774; https://doi.org/10.3390/microorganisms8111774 - 11 Nov 2020
Cited by 5 | Viewed by 2223
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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17 pages, 5572 KiB  
Article
Aestipascuomyces dupliciliberans gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca
Microorganisms 2020, 8(11), 1734; https://doi.org/10.3390/microorganisms8111734 - 05 Nov 2020
Cited by 24 | Viewed by 2909
Abstract
We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming [...] Read more.
We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming medium-sized (2–5 mm) white filamentous colonies with a white center of sporangia, on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7–20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described anaerobic gut fungal strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharide substrates as the sole carbon source. Phylogenetic assessment based on the D1–D2 28S large rRNA gene subunit (D1–D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1–D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the OrpinomycesNeocallimastixPecoramycesFeramycesGhazallamyces supragenus group. D1–D2 LSU and ITS-1 sequences recovered from the obtained isolates were either identical or displayed extremely high sequence similarity to sequences recovered from the same aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered preferably in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for “summer pasture”), and a new species, A. dupliciliberans. The type strain is Aestipascuomycesdupliciliberans strain R4. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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17 pages, 1899 KiB  
Article
Interrelationships of Fiber-Associated Anaerobic Fungi and Bacterial Communities in the Rumen of Bloated Cattle Grazing Alfalfa
Microorganisms 2020, 8(10), 1543; https://doi.org/10.3390/microorganisms8101543 - 07 Oct 2020
Cited by 12 | Viewed by 2144
Abstract
Frothy bloat is major digestive disorder of cattle grazing alfalfa pastures. Among the many factors identified to contribute to the development of frothy bloat, the disruption of rumen microbiota appears to be of central importance. Anaerobic rumen fungi (ARF) play an important role [...] Read more.
Frothy bloat is major digestive disorder of cattle grazing alfalfa pastures. Among the many factors identified to contribute to the development of frothy bloat, the disruption of rumen microbiota appears to be of central importance. Anaerobic rumen fungi (ARF) play an important role in sequential breakdown and fermentation of plant polysaccharides and promote the physical disruption of plant cell walls. In the present study, we investigated the dynamics of ARF during the development of alfalfa-induced frothy bloat and in response to bloat preventive treatments. By sequencing the internal transcribed spacer (ITS1) region of metagenomic DNA from the solid fraction of rumen contents, we were able to identify eight distinct genera of ARF, including Neocallimastix, Caecomyces, Orpinomyces, Piromyces, Cyllamyces, Anaeromyces, Buwchfawromyces, and unclassified Neocallimastigaceae. Overall, transition of steers from a baseline hay diet to alfalfa pastures was associated with drastic changes in the composition of the fungal community, but the overall composition of ARF did not differ (p > 0.05) among bloated and non-bloated steers. A correlation network analysis of the proportion of ARF and ruminal bacterial communities identified hub fungal species that were negatively correlated with several bacterial species, suggesting the presence of inter-kingdom competition among these rumen microorganisms. Interestingly, the number of negative correlations among ARF and bacteria decreased with frothy bloat, indicating a potential disruption of normal microbial profiles within a bloated rumen ecosystem. A better understanding of fungal-bacterial interactions that differ among bloated and non-bloated rumen ecosystem could advance our understanding of the etiology of frothy bloat. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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Review

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28 pages, 2154 KiB  
Review
The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production
Microorganisms 2021, 9(4), 694; https://doi.org/10.3390/microorganisms9040694 - 27 Mar 2021
Cited by 32 | Viewed by 6161
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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17 pages, 1264 KiB  
Review
Interactions between Anaerobic Fungi and Methanogens in the Rumen and Their Biotechnological Potential in Biogas Production from Lignocellulosic Materials
Microorganisms 2021, 9(1), 190; https://doi.org/10.3390/microorganisms9010190 - 17 Jan 2021
Cited by 30 | Viewed by 6626
Abstract
Anaerobic fungi in the digestive tract of herbivores are one of the critical types of fiber-degrading microorganisms present in the rumen. They degrade lignocellulosic materials using unique rhizoid structures and a diverse range of fiber-degrading enzymes, producing metabolic products such as H2 [...] Read more.
Anaerobic fungi in the digestive tract of herbivores are one of the critical types of fiber-degrading microorganisms present in the rumen. They degrade lignocellulosic materials using unique rhizoid structures and a diverse range of fiber-degrading enzymes, producing metabolic products such as H2/CO2, formate, lactate, acetate, and ethanol. Methanogens in the rumen utilize some of these products (e.g., H2 and formate) to produce methane. An investigation of the interactions between anaerobic fungi and methanogens is helpful as it provides valuable insight into the microbial interactions within the rumen. During the last few decades, research has demonstrated that anaerobic fungi stimulate the growth of methanogens and maintain methanogenic diversity. Meanwhile, methanogens increase the fiber-degrading capability of anaerobic fungi and stimulate metabolic pathways in the fungal hydrogenosome. The ability of co-cultures of anaerobic fungi and methanogens to degrade fiber and produce methane could potentially be a valuable method for the degradation of lignocellulosic materials and methane production. Full article
(This article belongs to the Special Issue Unleashing the Hidden Potential of Anaerobic Fungi)
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