Anaerobic Fungi: Comparison
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Anaerobic fungi, though low in abundance in rumen, play an important role in the degradation of forage for herbivores. Anaerobic fungi have been found in almost all animals that ferment in the predigestive tract, including ruminants (e.g., Bovidae, Cervidae), pseudoruminants (e.g., hippos, camels, llamas, alpacas), and nonruminants (e.g., wallabies). They are also found in many postdigestive tract fermenters that digest plant tissues in the cecum and large intestine (e.g., elephants, horses, and rhinoceroses) and in some large herbivore rodents (e.g., long-eared guinea pigs). It appears that the establishment of anaerobic fungi in the alimentary canal of herbivores may be attributed to the complex and distinct chamber with a relatively neutral pH in the digestive tract and a long lag time in the digestive process for the ingested plant tissue, which is conducive to the growth and activity of anaerobic fungi. Therefore, anaerobic fungi, which appear in the digestive tract of these herbivores, especially in those that take in a lot of roughage, must have some unique reasons and advantages to exist in such a complex environment. 

  • anaerobic fungi
  • classification
  • fiber degradation

1. Classification of Anaerobic Fungi

Early after the discovery of anaerobic fungi, they were considered to be protozoa with flagella due to their zoospores

1. Classification of Anaerobic Fungi

Early after the discovery of anaerobic fungi, they were considered to be protozoa with flagella due to their zoospores
[1]
. In the 1970s, Orpin suggested that these flagellates were actually the transmission stage of fungal zoospores
[2]
, and that chitin components in their cell walls
[3]
further confirmed their correct location in the fungal kingdom. Since then, a series of related experiments have established the classification system of the lignocellulosic-degrading fungi and the corresponding functional mechanism.
Anaerobic fungi belong to the fungi kingdom, phylum Neocallimastigomycota, order Neocallimastigales, and family Neocallimastigaceae. Up to now, 20 culturable genera of anaerobic fungi have been reported, containing Neocallimastix
[4]
, Caecomyces
[5]
, Orpinomyces
[6]
, Piromyces
[6]
, Anaeromyces
[7]
, Cyllamyces
[8]
, Buwchfawromyces
[9]
, Oontomyces
[10]
, Pecoramyces
[11]
, Feramyces
[12]
, Liebetanzomyces
[13]
, Agriosomyces
[14]
, Aklioshbomyces
[14]
, Capellomyces
[14]
, Ghazallomyces
[14]
, Joblinomyces
[14]
, Khoyollomyces
[14]
, Tahromyces
[14]
, Aestipascuomyces
[15]
and Paucimyces
[16]
. These isolated and cultured genera, however, only occupy a part of the natural anaerobic fungal communities, as revealed by a large and increasing number of sequences in public databases analyzed by a variety of molecular analysis tools
[17][18]
. At first, the classification of anaerobic fungi mainly depended on their morphological characteristics, such as monoflagellate fungi with less than four flagella on spores and polyflagellate fungi with more flagella, monocentric fungi with nuclei only in sporangia and polycentric fungi with nuclei in both sporangia and rhizoid system, and fungi with filamentous or bulbous rhizoid. However, when the nutritional environment changes, these morphological characteristics of anaerobic fungi also change. At the same time, morphological identification relies on the anaerobic cultivation, which identifies culturable species with a very limited range and gives rise to the great controversy and challenges of the morphological identification of anaerobic fungi. The development of molecular technology has broadened insight into microbial diversity and provided many practical approaches for the isolation and identification of anaerobic fungi.
The 18S rRNA gene sequence analysis technique was first used as a method for the evolutionary classification of anaerobic fungi. Bowman et al. used this technique to determine the location of anaerobic fungi in the class Chytridiomycetes
[19]
. Then Li and Heath believed that anaerobic fungi should be classified into the order Neocallimasticales, according to the 18S rRNA sequence structure
[20]
. More and more 18S rRNA gene sequences of anaerobic fungi have been uploaded to the gene database, and these sequences in the small ribosomal subunit (SSU) is highly conserved in Neocallimastigomycota, so its classification at the genus level is not very accurate and has certain limitations
[21][22]
. The internal transcribed spacer (ITS) is now becoming a commonly used fungal molecular marker, which consists of ITS1 (between 18S rRNA and 5.8S rRNA) and ITS2 (between 5.8S rRNA and 28S rRNA) in rRNA. ITS is relatively stable in the evolutionary process of fungi, and the hypervariable region of ITS has a greater relative change compared with 18S rRNA, so it is more suitable for studying the evolutionary relationship between fungi. ITS1 has been widely used to compare and classify anaerobic fungi of different species
[23][24][25]
. Although the ITS1 region of anaerobic fungi can be used for environmental sample analysis, the accuracy of this method is affected by the sample community composition. In addition, the fuzziness of sequence annotation in pure culture caused by the heterogeneity of ITS1 reinforces the limitations of the ITS1 region in the classification of anaerobic fungi
[26]
. Except ITS1, some researchers use the large ribosomal subunit’s (LSU) D1/D2 region as a symbol of the classification of anaerobic fungi, because these areas between different genera and species of fungi have more stable variation
[27]
. Compared with the method ITS1, LSU alignment for anaerobic fungal classification is more conserved due to the much lower degree of heterogeneity between the sequences and much less frequent presence of large insertions/deletions in some sequences. This makes LSU phylogeny avoid a misleading assignment owing to complicated variation, especially excessive intragenomic variation, which always leads to the ambiguous ITS1 alignment. For specific detection and phylogenetic placement of anaerobic fungi, the LSU method targeting the 28S rRNA gene appears to constitute a consistent and more reliable phylogenetic barcode
[28]
. The combination of ITS1 and LSU for the identification of anaerobic fungi can compensate for each other’s limitations, and for some species still in dispute, another molecular marker may need to be developed to determine their location.
Although there has been a rapid progress of molecular biology, which has promoted the development of anaerobic fungal genomics, it is a challenge to conduct the sequencing and identification and accurate classification of anaerobic fungi due to the high A-T content (~80%)
[29]
in their genome. So far, whole-genome sequencing has been published (
Table 1
) only for 11 species of 20 genera. More suitable molecular techniques should be developed to address these challenges.
Table 1.
The anaerobic fungal isolates (11) that have been genome-annotated in recent years.
Organism Assembly Length Genes Count Isolation Source Sample Published
Piromyces sp. UH3-1 84,096,456 16,867 Donkey Feces -
Piromyces sp. E2 71,019,055 14,648 Elephant Feces [30]
Piromyces finnis 56,455,805 10,992 Horse Feces [30]
Caecomyces churrovis A 165,495,782 15,009 Sheep Feces [31]
Anaeromyces robustus 71,685,009 12,832 Sheep Feces [30]
Neocallimastix sp. Gf-Ma3-1 209,503,801 28,646 Giraffe Feces -
Neocallimastix sp. WI3-B 206,810,295 28,960 Wildebeest Feces -
Neocallimastix lanati 200,974,851 27,677 Sheep Feces [32]
Neocallimastix californiae G1 193,032,486 20,219 Goat Feces [30]
Pecoramyces ruminantium C1A 100,954,185 18,936 Angus steer Feces [33]
Pecoramyces sp. F1 106,834,627 17,740 Goat Rumen sample [34]
Piromyces sp. UH3-1, Neocallimastix sp. Gf-Ma3-1 and Neocallimastix sp. WI3-B are three strains recorded in JGI’s MycoCosm (
(accessed on 10 December 2021)), but no references were found. All genome data can be downloaded at JGI (
https://genome.jgi.doe.gov/portal/neocallimastigomycetes/neocallimastigomycetes.download.html (accessed on 10 December 2021)).

2. Digestion of Plant Fiber by Anaerobic Fungi

There are a lot of lignocellulolytic bacteria in rumen. These bacteria secrete carbohydrate-active enzymes (CAZymes) to help digest complex and recalcitrant lignocellulose material of roughage in the host. In comparison with bacteria, anaerobic fungi not only possess physical penetration effect on plant tissue, but also secret a group of highly specialized CAZymes, including GH10, GH11, GH6, GH45, GH5, GH43, CE1, CE4, and and so on with higher abundance than bacteria during the enzymatic process
(accessed on 10 December 2021)).

2. Digestion of Plant Fiber by Anaerobic Fungi

There are a lot of lignocellulolytic bacteria in rumen. These bacteria secrete carbohydrate-active enzymes (CAZymes) to help digest complex and recalcitrant lignocellulose material of roughage in the host. In comparison with bacteria, anaerobic fungi not only possess physical penetration effect on plant tissue, but also secret a group of highly specialized CAZymes, including GH10, GH11, GH6, GH45, GH5, GH43, CE1, CE4, and and so on with higher abundance than bacteria during the enzymatic process
[35].

2.1. Physical Degradation with Fungal Rhizoids

Anaerobic fungi use flagella as their locomotor organ, and the chemotaxis of moving fungal zoospores to soluble sugars enables them to move quickly ahead of other rumen microorganisms and colonize on the ingested plant tissue
.

2.1. Physical Degradation with Fungal Rhizoids

Anaerobic fungi use flagella as their locomotor organ, and the chemotaxis of moving fungal zoospores to soluble sugars enables them to move quickly ahead of other rumen microorganisms and colonize on the ingested plant tissue
[36]
. It may provide a unique advantage for fewer anaerobic fungi to compete for nutrients in the complex rumen environment
[37]
. When they touch the surface of the plant, the zoospores spread out their flagella to form cysts. During the formation and growth of cysts, the rhizoids originate from the side of the cell opposite the insertion of the flagella, and is polar or lateral in position
[2]
, and these develop into a highly branched rhizoid system. The rhizoid system of the anaerobic fungi then penetrates the plant tissue and destroys its structure. This first step is physical degradation, which exposes a larger area for interaction between digestive enzymes and fibrous tissue, and releases the plant cell contents for utilization by itself (fungi) and other rumen microorganisms. With the invasion of the rhizoid system, a series of CAZymes are secreted to release cellulose, hemicellulose, and oligosaccharides from lignocellulose and convert these polymers into soluble sugars. These sugars could be used by hosts and microbiota within the system
[29][17][38][39]
. The important developmental stages of anaerobic fungi and their physical digestion of feedstuff can be clearly observed under a microscope (
Figure 1
).
/media/item_content/202204/624cfc5e1a954jof-08-00338-g001.png
Figure 1.
Anaerobic fungus Pecoramyces sp. F1 under the phase contrast microscope. The anaerobic fungus Pecoramyces sp. F1 undergoes the growth stages of zoospore attachment (
A
), rhizoid growth and penetration into the rice straw surface (
B
–
F
), and sporangium development and maturation (
C
–
E).

2.2. Digestion by Diverse Plant Fiber-Degrading Enzymes

The efficient degradation of forage by anaerobic fungi is also attributed to the complete variety of carbohydrate-active enzymes (CAZymes), including glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyl transferases (GTs). Carbohydrate-binding modules (CBMs) and less studied auxiliary activities (AAs) are accessories that are functionally related to CAZymes. Genome information of some anaerobic fungal strains that have been genetically annotated showed the presence of rich CAZymes, CBMs, and AAs of different types (
).

2.2. Digestion by Diverse Plant Fiber-Degrading Enzymes

The efficient degradation of forage by anaerobic fungi is also attributed to the complete variety of carbohydrate-active enzymes (CAZymes), including glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyl transferases (GTs). Carbohydrate-binding modules (CBMs) and less studied auxiliary activities (AAs) are accessories that are functionally related to CAZymes. Genome information of some anaerobic fungal strains that have been genetically annotated showed the presence of rich CAZymes, CBMs, and AAs of different types (
Figure 2
). Details on activities of these enzymes can be found in another article
[35]
. The most diverse group of CAZymes in the anaerobic fungi are the GHs
[35]
. Enzymes hydrolyzing cellulose, hemicellulose, pectin, and other plant wall polysaccharides constitute a large group of GHs. Among these enzymes, cellulases and hemicellulases are the main ones.
/media/item_content/202204/624cfc70400b9jof-08-00338-g002.png
Figure 2.
Genome-annotated information of anaerobic fungi. Anaerobic fungi strains that have been genome-annotated (Caecomyces churrovis A, Anaeromyces robustus, Neocallimastix californiae G1, Neocallimastix lanati, Neocallimastix sp. Gf-Ma3-1, Neocallimastix sp. WI3-B, Orpinomyces sp., Piromyces finnis, Piromyces sp. E2, Piromyces sp. UH3-1) can be found in JGI’s MycoCosm (
(accessed on 10 December 2021)), and the results of their annotation in the CAZymes database are included. The genome of Pecoramyces sp. F1 was uploaded to dbCAN2
[40]
for online annotation, and at the same time, BLAST
[41]
was used for the annotation of gene models against CAZymes database too. The data of AAs presented in the annotation results of Pecoramyces sp. F1 but that of other 10 strains cannot be found in JGI’s MycoCosm (
https://mycocosm.jgi.doe.gov/mycocosm/annotations/browser/cazy/summary;pEimlQ?p=neocallimastigomycetes (accessed on 10 December 2021)), so the data of AAs are not shown. dbCAN2 and BLAST results were combined to get the Pecoramyces sp. F1 genome annotation results in the figure.
(accessed on 10 December 2021)), so the data of AAs are not shown. dbCAN2 and BLAST results were combined to get the Pecoramyces sp. F1 genome annotation results in the figure.

Cellulase catalyzes the hydrolysis of β-1, 4-glucosidase in cellulose. Three types of cellulases (endoglucanase, exoglucanase, and β-glucosidase) that have been reported in anaerobic fungi together hydrolyze cellulose and release glucose[42]. Endoglucanase is the most important component of the cellulase system, which can cleave the glucan chain inside the cellulose. High levels of endoglucanase were found in the culture supernatants of Neocallimastix frontalis[43][44], Piromyces communis[45], and Orpinomyces Orpinomyces sp.[46]. Endoglucanase production was maximum when the fungi were grown on cellulose, whereas synthesis was totally repressed by the addition of glucose, indicating that the enzyme was subject to regulation [43]. Exoglucanase cleaves cellobiose units, the building blocks of cellulose, from the ends of the cellulose chain. Exoglucanase active against microcrystalline cellulose was also detected, but at lower levels than endoglucanase[43][46]. In addition, β-glucosidase cleaves cellobiose, which is a potent inhibitor of the former two enzymes, to yield glucose[47]. Previous studies have classified that the glycoside hydrolase enzyme classes GH1, GH2, GH3, GH5, GH6, GH8, GH9, GH38, GH45, GH48, and GH74 encode for cellulases[48]. Enzymes in different GHs have a different fiber hydrolysis activity according to their structure. The expression of these classes of enzymes varies according to fungal species and provided growth substrates. GH6 and GH5 were the two highest-expressed families in the Pecoramyces sp. F1 according to its transcriptome analysis, whereas GH1 and GH33 were the lowest-expressed ones[49]. In the genome of Pecoramyces ruminantium C1A, GH45 and GH48 were highly expressed with glucose as substrate, while the expression of GH6 was dramatically improved by corn stover[50].

Hemicellulose is the main component of the plant primary wall. It is a heteropolysaccharide containing xylan, glucuronoxylan, arabinoxylan, glucomannan, galactomannan, and xyloglucan[51][52]. The component monosaccharides are connected to each other, forming a hard part of the cell wall to prevent microorganisms from using the plant cell contents. The hemicellulases secreted by anaerobic fungi include xylanases, mannanases, galactanases, β-glucanases and so on, which can degrade hemicellulose into various oligomers and monosaccharides. Due to xylan being the basic polymeric compound of hemicellulose and the high hydrolase activity of xylanases, xylanases are the most studied enzymes to date among the hemicellulose hydrolases from anaerobic fungi[53][54][55]. Heterologous expression of these enzymes has also been developed. Xue et al. isolated a Neocallimastir patricianun xylanase cDNA and engineered it for heterologous expression in Escherichia coli (E. coli). The modified xylanase produced in E. coli had a specific activity of 1229 U/mg protein at pH 7 and 50 °C, without purification[56]. The genes encoding xylanases from Neocallimastix sp. GMLF2[57], Orpinomyces sp. Strain 2, and Neocallimastix frontalis[58] were also cloned into E. coli, and a high expression was obtained. In addition to E. coli, Hypocrea sp.[59], Kluyveromyces sp., and Pichia sp.[58] may also be ideal heterologous expression vectors for xylanases from anaerobic fungi. Successful development of heterologous production technologies for the enzymes from anaerobic fungi constitutes a significant advancement in applying this promising source of genes towards lignocellulose bioconversion. The glycoside hydrolase classes GH10, GH11, GH30, GH31, GH38, GH39, GH43, GH47, GH53, and GH115 encode for hemicellulases, respectively[48]. Xylanases are representative enzymes of GH10 and GH11. The former has a relatively high molecular weight, while the latter has a lower molecular weight.

In addition to the free enzymes secreted outside the cell, anaerobic fungi also secrete a number of large (MDa) multiprotein cellulolytic complexes, known as cellulosomes, which can degrade crude fibers more completely and efficiently. Although most studies have reported the function of cellulosomes in anaerobic microorganisms, such as anaerobic fungi, and the bacteria Clostridium thermocellum and Ruminococcus albus, there have also been reports on the discovery of cellulosomes in aerobic bacteria[60]. Cellulosomes include carbohydrate-binding domains, noncatalytic protein domains, and many glycosyl hydrolases (cellulases, hemicellulases, pectin enzymes, chitinases, and other enzymes). The assembly of cellulosomes involves interactions between dockerin domains on different catalytic enzyme subunits and cohesin domain on noncatalytic scaffolding, and the specific interactions between them allow various enzyme proteins to bind stably in this supramolecular structure. Scaffold proteins and some enzymes contain CBMs, which promote the binding of the cellulosome enzyme to substrate cellulose. Cellulosomes are attached to the cell surface by additional anchoring domains by noncovalent bonding[61]. Many GHs of anaerobic fungi are involved in the formation of cellulosomes. It was reported that anaerobic fungal cellulosomes exhibit additional 13% higher GH activity due to the presence of GH3, GH6, and GH45 compared with bacterial cellulosomes; especially the supplementary β-glucosidase conferred by GH3 activity empowers fungal cellulosomes in converting cellulose to single simple sugars (monosaccharides) when compared with low-molecular-weight oligosaccharide-generating bacterial cellulosomes[30][62]. Because of the powerful fiber degradation capability of cellulosomes, better than commercial preparations containing noncomplexed enzymes, the efficient degradation of cheap fibrous materials is possible if the modified cellulosome genes can be inserted into appropriate host cells and expressed using genetic engineering techniques.

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