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The equine gastrointestinal tract is a self-sufficient fermentation system, housing a complex microbial consortium that acts synergistically and independently to break down complex lignocellulolytic material that enters the equine gut. Despite being strict herbivores, equids such as horses and zebras lack the diversity of enzymes needed to completely break down plant tissue, instead relying on their resident microbes to carry out fibrolysis to yield vital energy sources such as short chain fatty acids. The bulk of equine digestion occurs in the large intestine, where digesta is fermented for 36–48 h through the synergistic activities of bacteria, fungi, and methanogenic archaea. Anaerobic gut dwell- ing bacteria and fungi break down complex plant polysaccharides through combined mechanical and enzymatic strategies, and notably possess some of the greatest diversity and repertoire of carbohydrate active enzymes among characterized microbes. In addition to the production of enzymes, some equid-isolated anaerobic fungi and bacteria have been shown to possess cellulosomes, powerful multi-enzyme complexes that further enhance break down. The activities of both anaerobic fungi and bacteria are further facilitated by facultatively aerobic yeasts and methanogenic archaea, who maintain an optimal environment for fibrolytic organisms, ultimately leading to increased fibrolytic microbial counts and heightened enzymatic activity. The unique interactions within the equine gut as well as the novel species and powerful mechanisms employed by these microbes makes the equine gut a valuable ecosystem to study fibrolytic functions within complex communities. This review outlines the primary taxa involved in fibre break down within the equine gut and further illuminates the enzymatic strategies and metabolic pathways used by these microbes. We discuss current methods used in analysing fibrolytic functions in complex microbial communities and propose a shift towards the development of functional assays to deepen our understanding of this unique ecosystem. Keywords Equine, Microbiome, Gastrointestinal tract, Fibre, CAZyme, Anaerobic fungi, Health plant based diet, equids lack the diversity of enzymes Introduction needed to break down complex plant polymers alone The equine hindgut is a complex naturally occurring fer - and have evolved symbiotic relationships with resident mentation system of powerful lignocellulolytic microbes. gut microorganisms to produce the enzymes that facili- ‘Fibre’ (complex plant polysaccharides) degradation is an tate plant matter degradation . These microbes fer - essential process in equine digestion, allowing the libera- ment complex carbohydrates in plant material into short tion of vital energy sources. Despite consuming a strictly chain fatty acids, contributing 60–70% of the horse’s daily energy requirements . An understanding of the equine hindgut microbial *Correspondence: Georgia Wunderlich ecosystem and the parameters that control and affect it email@example.com can greatly facilitate our understanding of host-microbe Tasmanian Institute of Agriculture, University of Tasmania, Hobart, interactions and how we can optimise the functionality of Australia Quantal Bioscience Pty Ltd, Castle Hill, Australia resident microbes and ultimately maximise energy yield within this environment. Our understanding of the role © The Author(s) 2023. 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Animal Microbiome (2023) 5:3 Page 2 of 17 of bacteria in hindgut fermentation is progressing [3–8], diet, supplements, coprophagy (faecal consumption) however, knowledge of the function of other microbial or other methods of ingestion [14–16]. Studies on the communities, including archaea and fungi, in the hind- meconium (first defecation) of newborn foals have found gut is still lagging . This review outlines the fibrolytic that their early GIT microbiome largely reflects bacteria microbial community composition of the equine hindgut found in the maternal milk  with other studies show- and elucidates the enzymatic processes and metabolic ing the equine microbiome generally begins to stabilise pathways that allow the break down of complex poly- between one and two months of age . A database has saccharides, as well as detailing current and future tech- recently been compiled to analyse taxon-associated host niques to assist further understanding of the mechanisms phenotypes and is available at http:// addag ma. omics bio. and functional genes underlining these processes. info/ to allow users to make biologically relevant que- ries about microbial related trends in equine health and The equine digestive habitat disease . The database summarises experimental Anatomy of the equine gastrointestinal tract observations found between domestic animals and their The majority of studies undertaken on herbivore diges - gut microbiota, however also highlights the lack of stud- tive systems, and particularly the microbial communities ies assessing these relationships in equids, with studies residing in them, have been done in ruminants, specifi - related to horse microbial phenotypes being significantly cally bovine and ovine. The horse gut shares several simi - fewer than those of cattle, pigs and chickens . The larities with the ruminant gut, having combined caecal entire equine GIT can contain up to 10 bacterial cells and colonic regions, with both being heavily dependent , and studies have reported as many as 10 fungal zoo- on their gut microbiota for digestion and nutrition . spores/mL of caecal content , although these num- Horses are monogastric herbivores, however, and do not bers do not necessarily equate to their functional value regurgitate digesta for further break down like foregut in the gut. The most important end product of fermenta - fermenting ruminants, having a greater dependence on tive processes carried out by these microbes are volatile resident hindgut microbes for fermentation and digestion fatty acids, such as propionate, acetate and butyrate   which are vital energy sources for horses, as well as car- The equine gastrointestinal tract (GIT) is aerobic to bon dioxide, water, methane, vitamins and several amino anaerobic from anterior to posterior, due to the intake acids . of oxygen with feeding and the subsequent utilization of Fibre degradation in the equine gut is primarily carried most of this oxygen by aerobic fermenters prior to reach- out by anaerobic bacteria and fungi, facilitated by facul- ing the hindgut . The horse’s stomach is the smallest of tatively aerobic yeasts and methanogenic archaea . any livestock or domestic animal relative to its size, hav- For many years protozoa were additionally considered ing a capacity of only 9–18 L . Most food degradation contributors to fibre degradation in the equine gut due to takes place in the large intestine which makes up over their ability to rapidly adhere to and colonize plant tis- 60% of the equine GIT. Upon entry to the large intestine, sue [23, 24]. An older study  showed their potential 85–95% of cell wall derived carbohydrates are undigested to enhance bacterial degradation of pectin and hemicel- . Digesta enters the large intestine approximately 3 h luloses, particularly arabinogalactan and galactomannan after feeding and is fermented for 36–48 h in the caecum components . However a later investigation found . The caecum sits at a pH of 6.3–7.5, ideal for the little impact of these microbes on plant digestion and growth of a plethora of anaerobic bacteria, fungi and pro- fibrolysis, and concluded that they have a minimal role in tozoa . Additionally, neurological signalling during directly degrading plant matter . Bacteriophages have feeding times trigger the caecum to increase in capacity also been suggested to influence the fitness of intestinal and mobility to enhance microbial-digesta interactions, cellulolytic bacteria and support colonisation, although allowing microbes to efficiently degrade difficult plant do not have a direct role in fibre break down . biomass and subsequently synthesise vital energy sources for the animal . Diet Being herbivores, equids source their nutrition from Equine gut microbiome plants, either through wild forages or commercial feeds. The dependence of equids on their gut microbiota and Plant cell walls are composed of lignocellulose, a struc- the consequent importance of this population has led ture made of two carbohydrate polymers, cellulose and to the viewing of the host and its microbes as one unit hemicellulose, and an aromatic heteropolymer, lignin, to when evaluating health and host phenotype, referred to bind the polysaccharides together (relative abundances as the ‘holobiont’ (a host and its microbiota) . Micro- of approximately 45%, 30% and 25% respectively) (see bial colonization of the equine gut is generally through Fig. 1) . W underlich et al. Animal Microbiome (2023) 5:3 Page 3 of 17 Fig. 1 Structure of the main components of plant biomass (cellulose, hemicellulose, and lignin). All components contain amorphous areas and variable structures and will not always present as the structures depicted above. (Adapted from ). Figure made in BioRender Cellulose, the major component of plant biomass, is or hexoses, interconnected by covalent hydrogen bonds one of the most abundant polysaccharides on earth . . The main structures of hemicellulose are xylan, Cellulose is composed of closely stacked disaccharide cel- xyloglucan, and galactomannan which vary in their struc- lobiose fibres linked by β (1–4)-glycosidic bonds, organ - ture and makeup depending on their respective sugars. ised in crystalline structures with scattered amorphous Sugars in hemicellulose are linked similarly to cellobi- areas. Despite being a relatively simple compound, the ose fibres by β (1–4)-glycosidic bonds and, occasion - proximity of these fibres to each other makes access - ally, β (1–3)-glycosidic bonds . Finally, lignin is an ing the bio-nutrients of cellulose difficult. Hemicel - amorphous heterogenous polymer composed of three lulose molecules are the second most abundant plant aromatic alcohols . The quantity and distribution of polysaccharide and present as polymers of pentoses and/ these aromatic alcohols varies between plant species. Wunderlich et al. Animal Microbiome (2023) 5:3 Page 4 of 17 Lignin is associated with hemicellulose and cellulose by components of lignocellulose. Carbohydrate degrada- ester and hydrogen bonds. It is these bonds between the tion involves two steps, the first being hydrolysis of plant different structural components of plant biomass, as well polysaccharides, and the second being the fermentation as the rigidity and complexity of individual components of the resulting simple sugars into short chain fatty acids that create the overall resistance to degradation of ligno- . cellulose complexes. ‘CAZymes’ are carbohydrate active enzymes which cat- Unsurprisingly, the composition of plant structure and alyze the break down and assembly of glycoconjungates contribution to herbivore feed has had a demonstrated and glycans . Currently there are six main CAZyme effect on the microbiome composition and overall fibro - classes, glycoside hydrolases (GHs), glycosyl transferases lytic capacity of the equine gut [14, 31–34]. Forages avail- (GTs), polysaccharide lyases (PLs), carbohydrate ester- able for grazing equids change substantially across the ases (CEs), carbohydrate binding modules (CBM) and globe and throughout the year, leading to corresponding auxiliary activities (AAs) which are redox enzymes that changes in feed nutrient value and digestibility. Gener- facilitate CAZyme function. These enzymes are classi - ally speaking, grasses grown during a rainy season are fied according to the different mechanisms in which they higher quality and more nutrient dense, while a dry sea- break down substrates, as well as their three-dimensional son brings increased lignin in plants, the least digestible folding structure characteristics and protein sequence fibre component of plants, and decreased nitrogenous similarities . An overview of different CAZymes is content . Feeds harvested during dry climatic condi- available at http:// www. cazy. org/ which has recently been tions generally lead to reduced fibre utilization due to the updated and reviewed . low nitrogenous content of this feed meaning there are Endoglucanases, exoglucanases and β-glucosidases less nitrogenous precursors for the synthesis of microbial are the three main cellulase groups that act synergisti- compounds such as enzymes . The effects of this may cally and simultaneously to break down cellulose using be partially overcome with nitrogenous dietary supple- hydrolysis . Endoglucanases target amorphous areas mentation which has been shown to stimulate gut fibro - of the crystalline cellulose matrix and cut into them, lytic activity and increase degradation of low quality fibre producing a chain end that exoglucanases are then able . to bind to and cleave, releasing the cellobiose fibres and Julliand and Grimm  provide an overview on the individual glucose monomers . Finally, the cellobiose impact of diet on the equine hindgut microbiome. Gen- is degraded to glucose monomers by β-glucosidases . erally, studies show diets high in starch have adverse While hydrolysing cellulases are the prominent degrad- effects on microbial populations in the equine gut, reduc - ers of cellulose, other cellulases exist which utilize other ing cellulolytic bacterial counts and overall fibrolytic modes of action, such as cellodextrinases and cellobiose capacity [34, 36, 37]. Such feed types are common among phosphorylases, which use phosphorylation-mediated domesticated pet horses in the form of pelleted horse cleavage, or oxidoreductases which use an oxidative feeds high in barley, oats and corn . Correspondingly, mode of action. Various microorganisms can produce these high concentrate diets can lead to an increase in cellulases, including bacteria, fungi, metazoan and some amylolytic bacteria, the primary one being Lactobacillus animals like termites, snails and crayfish [47–49]. species , overpopulation of which can lead to gut aci- Several enzymes are involved in the break down of dosis and ultimately colitis and laminitis [3, 39, 40]. High hemicellulose which act specifically on the different glycol fibre diets, in contrast, seem to maximise the fibrolytic units and glycosidic bonds. These hemicellulases include capacity of the equine gut, leading to increased cellulo- endo- and exo-β-glucanases and xylanases, polygalactu- lytic and xylanolytic bacterial counts [32, 41], as well as ronases, pectin methyl esterases, β-mannanases, feruloyl decreased concentrations of bacteria typically associated esterases, pectin and pectate lyases and arabinofuranosi- with laminitis induction, such as Lactobacillus . While dases . Hemicellulases are primarily produced by these studies highlight apparent trends between forage saprophytic microbes isolated from decaying plant and composition and microbial populations, other studies animal material . have suggested the reaction of the horse gut microbiome Current research on lignin degradation has focused to changes in plant structure to be largely individualised on the role of fungi in this process , however bacte- between horses, with implications on metabolic health ria have also been demonstrated to produce enzymes remaining to be elucidated . enabling lignin break down . The enzymes primarily responsible for lignin break down are laccases and perox- Fibre degrading enzymes idases and can be generally divided into two main groups; The complete break down of plant biomass requires lignin modifying enzymes and lignin degrading auxiliary the presence of multiple enzymes to target different enzymes, the latter of which are unable to degrade lignin W underlich et al. Animal Microbiome (2023) 5:3 Page 5 of 17 on their own but are necessary to complete the degra- as zooflagellates, early research on anaerobic fungi was dation process. These proteins primarily fall under the centralised around their role in breaking down feeds [65, ‘auxiliary activities’ family classification within the CAZy 66]. These fungi have since been isolated from diverse database. Lignin degradation is an oxidative process, and extreme environments, including in bedrock deep in therefore any microbes involved in this process within the earths biosphere . The uniqueness of this group the equine gut are likely rare and difficult to detect due of fungi stretches beyond their ability to survive under to the prominent anaerobicity of the equine hindgut . the anaerobic conditions of the equine gut, and has pro- Further culture-independent research is needed to fur- pelled research into understanding their taxonomy, enzy- ther elucidate microbial methods of lignin break down mology, morphology, and diversity in host animals. Hess within the equine GIT. et al. provides a comprehensive overview of past, present CAZymes are a growing focus in a range of research and future research on these distinctive organisms . fields due to their diverse industrial applications and All described anaerobic fungi are members of the phyla environmental distribution. Anaerobic gut dwelling bac- Neocallimastigomycota, which contains a single order teria and fungi possess some of the greatest diversity and (Neocallimastigales) and family (Neocallimastigaceae). repertoire of CAZymes amongst characterized microbes Within this family are 20 recognized genera, eleven of [55, 56], and the ecosystem of the equine may even which have only been characterised in the last five years serve as an environment for horizontal gene transfer of [68–71]. To date, at least six genera have been isolated CAZymes between organisms, as demonstrated in rumi- from the equine gut (Piromyces [72–79], Orpinomyces nant species . Comprehensive gene cataloguing of the [72, 80], Neocallimastix [72–74, 80], Anaeromyces [72, faeces from eleven endurance trained horses found 137 74, 80], Caecomyces [72–74, 77] and Khoyollomyces [68, different CAZymes through shotgun sequencing meth - 73, 80]), with several more uncharacterised species also ods, the majority (85.4%) belonging to glycoside hydro- being found with the development of next generation lase and polysaccharide lyase families , highlighting sequencing technologies [73, 74, 80]. A list of publicly the wealth of fibre degrading enzymes in the horse gut. available whole genomes of anaerobic fungi was com- The equine hindgut also houses microorganisms that piled by Hess et al.  and included one horse habituat- are exceptional because they can produce ‘cellulosomes’ ing organism, Piromyces finnis . which are multi-enzyme complexes made up of multi- Anaerobic fungi obtain energy from breaking down ple CAZymes. Cellulosomes co-ordinate lignocellulo- plant carbohydrates using cellulolytic enzymes, promi- lytic enzymes of similar functions to colocalise within nently CAZymes. An overview of CAZymes found in the complex for enhanced degradation . Celluloso- well-characterized equine microbes are shown in Table 1. mal enzymes are bound to a non-enzymatic membrane- The functional diversity of CAZymes in gut dwelling anchoring protein, a scaffoldin, via a modular dockerin fungi is impressive and far exceeds that of species cur- domain attached to the enzymes which interact and bind rently used in cellulolytic cocktails for biotechnological with cohesion proteins on the scaffoldin [60, 61], thus purposes, such as Aspergillus niger and Trichoderma resii creating dynamic and powerful catabolic complexes (see [59, 82]. In fact, early-branching anaerobic fungi encode Fig. 2). the largest number of biomass-degrading enzyme genes found in nature to date, with the genome of a strong Fungi plant-degrading anaerobic fungus typically harbouring Anaerobic fungi 200–300 CAZyme specific genes [44, 81]. This has been Anaerobic fungi are believed to be major contributors to attributed to early horizontal gene transfer of bacterial fibrolysis and hindgut fermentation , found to rep - hemicellulases to anaerobic fungi , providing anaero- resent at least 20% of microbial biomass in the rumen bic fungi a more diverse repertoire of enzymes compared (63). Anaerobic fungi are significantly better degrad - to later diverging fungi which lack substrate catabolism ers of plant cell walls compared to bacteria, due to their diversity . As such, anaerobic fungi have demon- larger repertoire of fibre degrading enzymes and cellu - strated efficient digestion of all major components of losomes and their ability to mechanically invade plant tis- plant wall material including cellulose, xylan and galacto- sue with penetrative hyphae [56, 62, 64]. Yet, this niche mannan . In ruminant GITs, Neocallimastigomycota group of microorganisms remains greatly understud- have been found to be responsible for the fermenta- ied within herbivore research. Neocallimastigomycota tion of 18–63% of untreated plant biomass [56, 84, 85] is the only described anaerobic fungi phylum and their despite only being approximately 8% of the gut microbi- production of lignocellulolytic enzymes are central to a ome biomass . In bovines, the removal of anaerobic range of agricultural, biogeochemical, and nutritional fungi through treatment with cycloheximide and tetro- processes . First identified in the herbivore system nasin was shown to reduce intake of low quality feed to Wunderlich et al. Animal Microbiome (2023) 5:3 Page 6 of 17 Fig. 2 The role of fungi in plant break down and metabolism. 1 Schematic diagram of the equine digestive system (red text indicates the foregut, blue text indicates the hindgut). The majority of hindgut digestion occurs in the caecum. 2 Plant matter in the caecum is invaded by penetrative hyphae of anaerobic fungi. 3 Overview of the enzymatic activity of anaerobic fungi (adapted from ). Anaerobic fungi degrade plant biomass within the equine caecum through several enzymatic strategies; free carbohydrate active enzymes (CAZymes), cell bound cellulosome complexes and free cellulosomes secreted by the cell. Cell bound cellulosome example given is of glycoside hydrolase 3 which converts cellulose to monosaccharide glucose molecules via β-glucoside activity. These glucose molecules can then be absorbed in the equine gut or enter the fungal metabolic pathway. 4 Example of energy metabolism of Piromyces sp. E2 (adapted from ). Glucose molecules enter the glycolysis pathway, the product of which are two pyruvate molecules which either enter a mixed acid fermentation in the cytosol, or the hydrogenosome for ATP generation. Major by-products of fungal energy are indicated by the thick orange arrows. Figure made in BioRender 70% . This is likely as the mechanical disruption and down of plants by anaerobic fungi including capturing subsequent enzymatic break down by anaerobic fungi of surface localised cellulosomes. Images revealed fungal would ordinarily speed up feed break down and allow rhizoids invasively covering grass particles, enhancing more rapid clearance of digesta [22, 87], underlining their surface coverage and access to carbon sources. Also vis- important role in the digestion of crude lignocellulose ible was hyphal penetration of plant substrate to access and subsequently host nutrition. trapped carbon . Hyphal tips, furthermore, have a In addition to their enzymatic potential, anaerobic high concentration of fibrolytic enzymes, the activity of fungi offer an added benefit in fibrolysis through mechan - which increase nutrient availability for other cellulolytic ical agitation and hyphal invasion of plant tissue . microbes including bacteria . Helium ion micrographs of the equine derived anaerobic The simple monomers from lignocellulose degrada - fungi P. finnis presented by Lillington et al. , dem- tion, such as glucose, are metabolised by fungi though a onstrated the dynamic mechanical and enzymatic break type of mixed acid fermentation (i.e., heterofermentative W underlich et al. Animal Microbiome (2023) 5:3 Page 7 of 17 Table 1 CAZyme families from prominent fibre degrading microbes isolated from the equine gut Numbers under taxa indicate CAZyme families found within the corresponding CAZy class in each taxon. Fungal CAZymes found are heterologously expressed within each genus. Where enzyme functionality has been characterized, the protein name is written in brackets after the CAZy family number. Fibrolytic activities in blue are involved in hemicellulose degradation, while activities in orange are primarily cellulose degrading. Note- this is not an exhaustive list of fibrolytic microbes found in the equine gut, but rather a summary of those for whom enzymology has been described *NC = CAZymes “non classified” CAZymes; i.e. not yet assigned to a family processes), where degradation of carbohydrates leads to identified. The composition of the mycobiome was found the production of hydrogen, carbon dioxide, formate, to have a higher association with diet than species types, acetate, succinate, and ethanol as by-products  (see (e.g., zebra, horse, donkey) , highlighting the impor- Fig. 2 for an overview of anaerobic fungi plant break tance of equine nutrition in mycobiome development. down and metabolism). The bulk of fungal metabolism Past and present investigations into the equine hindgut is conducted by hydrogenosomes, an evolved version of have revealed its distinction from other anaerobic fungi the membrane bound organelle mitochondria used by ecosystems, both functionally and taxonomically. Several respiration dependent organisms [21, 88, 89]. Rather than studies have revealed the presence and dominance of the carrying out oxidative phosphorylation, this structure genera Khoyollomyces (formally known as AL1), in the produces ATP through substrate-level phosphorylation equine hindgut, having been found almost exclusively in under anoxic conditions, producing hydrogen as an end- equines [39, 44, 53, 55]. The type species Khoyollomyces product . This hydrogen is considered vital for the ramosus (khyollo meaning horse, myces meaning fungi) growth of methanogenic archaea and bacteria , high- was initially cultivated from zebra and equine faeces lighting the importance of anaerobic fungi in the equine . Isolates of K. ramosus were observed to produce GIT microbial ecosystem. monoflagellated zoospores and develop highly branched Donkeys have been demonstrated to have increased rhizoids when encysted . The extensive rhizoidal fibre degradation abilities compared to horses  which branching of this species may explain its increased dis- is possibly explained by a study  comparing the ruption of plant tissue compared to other anaerobic fun- microbial populations of different Equidae species, which gal species [59, 83], however this is yet to be investigated. found donkeys to have a six-fold greater anaerobic fungal The recent cultivation and, therefore, characterization loads compared to horses, as determined using quanti- of this genus explains why its role in the equine gut, and tative polymerase chain reaction methods. Another key potentially novel enzymatic and metabolic pathways, are finding from that research was the strong correlation of currently unknown. diet with the types and concentrations of anaerobic fungi Wunderlich et al. Animal Microbiome (2023) 5:3 Page 8 of 17 The adaptation of anaerobic fungi to the gut environ - occasions, including from the horse [99, 100]. However, ment of horses has been further emphasised through it was initially thought that the fungi colonized faecal metabolic studies into equine derived fungi. Perhaps content post defecation and consequently thrived in the the most well documented genera of anaerobic fungi warm conditions. in herbivorous mammals is Piromyces spp., including Studies that uncovered this process [98, 99] noted that their production of highly effective cellulolytic enzymes. gut dwelling anaerobic fungi seemingly lack auxiliary Interestingly, metabolic differences have been reported activity 9 family of enzymes (“AA9”) including lytic poly- between strains of Piromyces isolated from equid and saccharide monooxygenases. These enzymes cleave cellu - rumen guts, with equine derived strains possessing lose fibres via C1 and C4 oxidation , making fibres higher fibre degrading capabilities and overall faster more readily digestible by other microbial groups. The growth rates . After 25 h of growth on soluble sug- study successfully isolated C1 and C4- oxidized cellulose ars, fungal biomass (mg/ mL) from equine derived strains from both horse faeces and directly from the horse stom- (ponies and donkeys) was three-fold higher than that ach and subsequently isolated three thermophilic fun- of rumen strains grown on the same substrate. Digesta gal species which were cultivated at 50 °C; Chaetomium spends a shorter amount of time in the equine caecum thermophilum, Thermoascus aurantiacus and Scyta - compared to the rumen foregut, and therefore equine- lidium thermophilum . All three species when iso- strains of Piromyces have likely adapted to induce plant lated directly from equine digesta were found to express cell wall break down at an increased rate . Of par- AA9 enzymes, confirming their role in cellulose cleavage ticular significance within the Piromyces genus is P. equi, within the equine stomach. The study went on to postu - a fungus isolated from equines that was found through late that these aerobic fungi grow in the anterior region protein and enzyme assays in conjunction with matrix- of the horse gut where oxygen is consumed, consequently assisted laser desorption/ionization time-of-flight mass facilitating the anaerobic conditions of the lower gut . spectrometry analysis, to possess a major exoglucanase- Notably, enzyme activity of thermophilic organisms is Cel6A. Cel6A is one of the most widely studied cellulo- often maximised at temperatures higher than that of the lytic enzymes as it is capable of fully digesting cellulose equine gut, and as such, it remains to be investigated how [92, 93], a key example of the powerful fibrolytic enzymes actively involved they are in plant break down within the at play in equine digestion. equine GIT. Several uncultivated anaerobic fungal clades have also been identified in the equine gut, including NG1, NG2, Facultatively aerobic yeasts NG3, NG5, NG7, DT1, KF1, SK1 and SK3 [73, 74, 80]. The role and presence of yeasts in the equine hindgut is However, many limitations currently exist in both tradi- even less explored than anerobic fungi, which are rarely tional and molecular identification of anaerobic fungi, reported as residents of this ecosystem. All yeasts belong including their stringent cultivation requirements, A-T to two phyla; Ascomycota and Basidiomycota within the and repeat rich genome, unknown ploidy, lack of a reli- Dikarya subkingdom . Yeasts are highly ubiquitous able DNA barcode and complex physiology . Addi- in the environment, found in a variety of habitats as tionally, reference sequences for these taxa are not well pathogens, transients, or symbionts [103, 104]. Although represented in publicly available databases for both their some yeast species enter the equine gut, they are often taxonomy and functionality. Tackling these technical considered non-functional transients in this eco-system, issues is essential to improving our understanding of the despite their ability to survive in this niche environment role these microbes play in fibrolysis. . Yeasts found within herbivore digestive tracts are not strictly anaerobic like their mould and bacterial Thermophilic fungi counterparts but instead considered ‘facultatively aero- Only recently has it been demonstrated that oxidative bic’, being able to survive with little to no oxygen . cleavage of cellulose occurs in the equine gut via aero- This is due to mitochondrial adaptations in the form of bic thermophilic fungi . Thermophiles are a type of deletions, mutations or duplications of mitochondrial extremophile microbe that can survive relatively high DNA or nuclear DNA involved in oxidative phosphoryla- temperatures, over 100 °C for some Eubacterial and tion . These species, often referred to in the litera - methanogenic Archean species [96, 97], or up to 60 °C for ture as ‘petites’, are respiration deficient but viable, and thermophilic fungi, the only eukaryotes demonstrated to unable to grow on non-fermentable substrates such as grow at such high temperatures . Thermophilic fungi, ethanol or glycerol . similar to anaerobic fungi, are a point of interest due to Much research on yeasts in herbivore digestion has their rapid growth rates and high cellulolytic activity been in insects, however the insect hindgut is more and have been isolated from herbivore faeces on several adapted to re-absorption of amino acids rather than plant W underlich et al. Animal Microbiome (2023) 5:3 Page 9 of 17 break down, and there is otherwise little overlap between current databases [127–130]. Through shotgun metagen - the GIT of these and horses. Some yeasts found in the omic sequencing, Gilory et al. , recovered 123 aforementioned studies include genera also isolated from metagenome assembled genomes (MAGs) belonging to the equine gut, including some Candida and Saccharo- archaea and bacteria from five horse faecal samples. The myces species [105, 107]. Yeasts isolated from the gut of bacteria detected were predominantly members of the insect herbivores, namely termites and beetles, have been phyla Proteobacteria, Firmicutes, Bacteroidetes and Act- shown to produce extracellular enzymes, particularly inobacteria, and collectively possessed a diverse array of xylan- and arabinogalactan-hydrolysing glycosidases, to polymer degrading enzymes . Each bacterial MAG break down hemicellulose and detoxify toxins in the her- possessed on average 69 different CAZymes with Bacte - bivore diet [108–110]. These findings, although not dem - roidota phyla members having the largest CAZyme rep- onstrated in equine derived yeasts, allude to the role of ertoire. The majority of reported CAZymes belonged to GIT yeasts in contributing to plant break down in con- the glycosyl hydrolase family (51%), indicating the potent junction with their other microbial counterparts. role of these microbes in the break down of complex car- bohydrates . Fibrolytic bacteria The enzymatic profiles of different microbial groups Bacteria are the most well documented microbial group within the equine gut typically shows trends of anaero- within the equine GIT ecosystem, and undoubtably have bic fungi being the primary degraders of cellulose, while important roles in fibre degradation and host health. fibrolytic bacteria target the degradation of hemicel - While the cellulase diversity of anaerobic fungi is greater, lulose. This functional specificity has particularly been cellulolytic bacteria are often found in higher concentra- demonstrated in the rumen. One such study assessed tions throughout the GIT and have faster growth rates, kingdom specific functionalities within the rumen micro - consequently often being found to have higher cellu- biome through placing nylan bags containing switch lase counts . Gut dwelling bacteria are well stud- grass directly into the rumen of two fistulated cows ied in mammalian hosts, having several proven roles in . Metatranscriptomics on the rumen incubated bags immunoregulation and other cell regulatory mechanisms revealed bacterial populations were primarily responsi- . Their fibrolytic role has been well established in ble for hemicellulose degradation, with the proteome of the rumen caecum [113–123] and to a lesser extent, the Fibrobacter succinogenes in particular containing a wealth equine hindgut [3–8]. of CAZymes from hemicellulose prominent families The functionality of gut dwelling bacteria can broadly (GH11, GH51 and GH94). The proteome and transcrip - be classified into proteolytic, lactate-using, glycolytic tome of rumen fungi however appeared better equipped and cellulolytic bacteria, the latter of which are mainly for the degradation of cellulose structures with enzymes composed of species from the phyla Actinobacteria, Fir- from the glycoside hydrolase family GH48 being the most micutes, Proteobacteria and Bacteroidetes [12, 124]. Few abundant CAZymes detected . cellulolytic bacteria have been isolated from the GIT of The enhanced hemicellulolytic catabolism of equine equines through culture dependent methods , possi- caecal bacteria has been further demonstrated through bly due to their slow growth rate and purported long lag their substantial growth on xylan rich media, the main phase to initiate substrate digestion . Additionally, carbohydrate component of hemicellulose, and subse- it is estimated that 33–80% of bacteria in the gut of her- quent poor colonization on cellulose rich media . The bivores are oxygen sensitive, including most cellulolytic xylanase activity of the dominant horse caecal isolate, bacteria  further challenging attempts to culture them Enterococcus casseliflavus, was also superior to that of and conduct functional evaluation. Current estimations isolates from buffalo and horse dung. The same study also of cellulolytic and hemicellulolytic bacterial populations found growth of an unidentified ligninolytic bacterium 4 8 in the equine hindgut range from 10 to 10 cells/ mL and on lignin. Other cellulolytic bacteria isolated from horse 6 8 10 to 10 cells/ mL respectively, showing their dynamic faeces with substantial hydrolytic capacity include Paeni- functionality in breaking down different plant wall com - bacillus polymyxa, Enterobacter cloacae and Escherichia ponents within the hindgut . coli, the former of which yielded the highest cellulolytic Few studies have undertaken enzymatic profiling of activity of those isolated . equine fibrolytic bacteria. Genome annotation is a com - Firmicutes are the largest phylum represented in the mon method for evaluating the enzyme richness of equine GIT, ranging from 40 to 90% in different sections microbial species, however, it is especially difficult for . Within this phylum is the class Clostridia which environmental and otherwise complex samples, due to contains the obligately anaerobic family Lachnospiraceae. the large diversified amount of data yielded from these Lachnospiraceae are regarded as core microbiome mem- analyses, and general absence of environmental species in bers across most animals [133–135], and are recognised Wunderlich et al. Animal Microbiome (2023) 5:3 Page 10 of 17 for their ability to degrade a variety of plant polysaccha- producing species suggests this is a mechanism likely rides , resulting in the production of short chain employed by equine cellulolytic bacteria as well . fatty acids such as butyrate which has a demonstrated Several studies looking into the occurrence of equine protective effect on colonocytes and serves as their main metabolic disorders analysed fluctuations on cellulolytic energy source . The families Ruminococcaceae and bacteria under different conditions [8, 38, 39]. A case Fibrobacteraceae are also members of Clostridia, and study on fifteen horses treated with different antibiot - while often only representing a small portion of the ics, showed a remarkable decrease in the presence of cel- equine microbiome, have been found as core members lulolytic bacteria (> 99% decrease), following treatment, in the equine gut [9, 138]. Ruminococcus and Fibrobacter through culture-based methods . After antibiotic with- are the two main genera involved in cellulose degrada- drawal, the levels of cellulolytic bacteria in treated horses tion in the equine gut, specifically, Ruminococcus albus, remained substantially lower than in control horses, and Ruminococcus flavefaciens and Fibrobacter succinogenes, continued to decrease for a week after withdrawal . and which are often used as markers for cellulolytic activ- Subsequently, this decrease in beneficial GIT bacteria ity in herbivore gut studies  (see Table 1 for an over- allowed colonization of potentially pathogenic bacteria, view of functional enzymes). including Salmonella and Clostridium difficile which are Ruminococci are one of few bacteria isolated from the common causes of diarrhea and other equine GIT disor- gut with the ability to produce cellulosomes [139, 140]. ders . A decrease in cellulolytic bacteria in the equine As previously mentioned, the use of functionally organ- large intestine was shown to correspond with a decrease izing CAZymes into a cellulosomal network greatly in the production of acetate, indicative of decreased fibre enhances the fibrolytic capacity of an organism. R. fla - degradation . vefaciens has frequently been identified as the main cel - lulolytic bacterial species in the equine gut, ranging in Microbial synergism concentrations throughout the lower GIT of between Microbial synergism is defined as the mutually benefi - 2 and 9.7% of overall bacterial populations [3, 141]. cial increase in the productivity or growth of a microbial Assembly of a cellulosomal complex from R. flavefaciens community resulting from the metabolic interactions of revealed a diversity of CAZyme family domains, includ- two or more microorganisms, to which their combined ing glycosyl hydrolase, carbohydrate esterase and pectate effect is greater than the sum of their separate abilities lyase binding domains, however the function of around [145, 146]. This is generally achieved through nutritional 30% of these proteins remained unknown . A unique interdependence, whereby one species can utilize the feature of the cellulosome of R. flavefaciens is its adap - products of another, minimizing by-products of the pro- tor scaffoldin, ‘ScaC’, which possesses the ability to bind ducer while providing nutrition to the feeder [147, 148]. to different dockerin groups and consequently modulate Fungi and bacteria both have independent, but syn- enzyme integration into the cellulosome complex based ergistic, fibre degrading roles in the equine hindgut, on functional needs . Furthermore, the cellulosome enhanced by their interactions with one another and of R. flavefaciens showed preferential recruitment and other microbes present. As previously described, anaer- appendage to hemicellulases, highlighting the prominent obic fungi are the primary colonizers of plant biomass role of bacteria in hemicellulose degradation . within the equine gut, their mechanical disruption of The ability of bacterial cellulosomes networks to tightly plant cuticles  enabling fibrolytic bacteria access to adhere to plant fibres within the gut has been recently plant fibre which they would not otherwise be able to fer - revealed , further enhancing microbe-substrate ment . The positive effects of anaerobic fungi on bac - interactions. Ruminococcus champanellensis, a cellulo- terial populations have particularly been demonstrated lytic bacterium isolated from the human gut, employs through administration of anaerobic fungal cultures as cellulosomes as a mechanism to anchor itself to plant feed additives. Paul et al. , fed cultures of the anaer- substrates and withstand the motility of the GIT . obic fungus Piromyces sp. FNG5 (previously isolated Through modelling and molecular dynamic simulations, from wild bull faeces) to buffaloes and showed significant researchers were able to show the cohesion-dockerin increases in cellulolytic and hemicellulolytic bacterial complex of these cellulosomes were able to regulate the counts, as well as increases in resident fungal popula- adhesion of bacteria to substrates under different envi - tions . Increases in fibrolytic microbial populations ronmental conditions such as pH and high stress, with was concomitant with increased volatile fatty acid pro- the protein bond becoming stronger as force increased duction and increased xylanase, cellulase, protease, and . No studies have yet reported the isolation of this acetyl and feruloyl esterase activities . This is con - Ruminoccous species from the equine gut, however sistent with other studies demonstrating that the addi- the presence of close relatives and other cellulosomal tion of fibrolytic fungi, to animal feed, increases bacterial W underlich et al. Animal Microbiome (2023) 5:3 Page 11 of 17 populations two-fold and subsequently enzyme activities belonging to CAZymes . DNA methylation is an epi- and feed digestibility within the gut [151, 152]. genetic process in which methyl groups are added to a Yeast supplementation has been shown to have ben- DNA molecule, which can change the activity of a DNA eficial effects on rumen bacterial populations through segment through state specific control of gene expression increased production of short chain fatty acids and vita- . Furthermore, co-culturing of the anaerobic fungi mins [153, 154], however results in equines have been and methanogen resulted in a proportional increase of less consistent. Garber, Hastie and Murray  have CAZyme gene expression, the majority of which were compiled a list of studies that use the yeast Saccharomy- either carbohydrate binding modules, fungal dockerin ces cerevisiae as a probiotic in equines. Of five studies domains or of unknown function . Some cellulases conducted, two recorded little to no effect on bacterial contain an active domain site for binding to carbohy- populations analysed from either the gut or faeces [155, drate binding modules , with those bound to a car- 156], and the other three noted variable effects [103– bohydrate binding module consistently demonstrating 105]. In response to yeast supplementation, one study enhanced cellulose attachment [169–171]. Carbohydrate noted decreased Streptococci in the colon and increased binding modules have diverse ligand specificity and can Lactobacillus in the caecum , while another study maintain enzyme–substrate proximity, ultimately leading recorded overall decreased Lactobacilli counts in faeces to prolonged and steady enzyme–substrate interactions . The fifth study recorded reduced F. succinogenes and increased enzyme concentrations on the polysac- populations in faeces but otherwise no effect on bacterial charide surface [169, 172]. These enhanced interactions populations or equine fibre digestibility . Ultimately can increase the hydrolytic activity of enzymes on soluble it remains unclear whether yeasts in the equine GIT can substrates , however deeper investigation is needed produce substantial changes in the digestibility of plant on their role in plant break down within an intestinal substrates. A deeper understanding of the role of yeasts setting. within the equine gut would greatly facilitate develop- ment of a more suitable feed supplement to assist plant Future directions: towards developing a functional matter break down. assay The symbiotic relationship between anaerobic fungi Understanding the composition and functionality of and methanogenic archaea has been a point of inter- fibrolytic microbial communities within the equine est in herbivore digestive systems , however little hindgut is of great importance in equine nutrition and work has been done on understanding this relationship to optimise energy yield from plant matter. Gastroin- within the equine gut, with archaea only being first iso - testinal disturbances induced by changes in the gastric lated from the equine gut in 1996 . The diversity of microbiome (‘dysbioses’), brought on by disease, change methanogenic archaea within the equine gut varies along of diet, antibiotic use, age or other factors, can result in the GIT and depending on host animal, with the horse compositional shifts of the equine microbiome, result- hindgut showing a predominance for Methanobacte- ing in fermentation dysfunction and ultimately metabolic rium-like sequences, and the donkey hindgut housing disorders . In the instance of the equine gut, identi- more Methanocorpusculum-like sequences . The fying microbes present within this environment and the production of methane by methanogenic archaea within repertoire of enzymes they possess can help researchers the herbivore gut is a by-product of fibre digestion [163, and veterinarians alike better understand the taxa associ- 164]. Hydrogen is produced by anaerobic fungi and bac- ated with a healthy or diseased animal, and on a larger teria and is reduced to methane by methanogens, allow- scale, can help researchers underpin the important taxa ing maintenance of low hydrogen levels within the gut, to involved in complex fibre degradation. consequently maintain thermodynamic requirements for It is evident through elucidation of the fibrolytic capac - anaerobic fermentation . ity of the equine gut that more detailed investigations of In addition to the beneficial waste removal by metha - this community require a holistic understanding of both nogenic archaea, their interactions with anaerobic fungi taxonomy and functionality, with an emphasis on enzy- have also demonstrated increased transcription of impor- mology of particular importance in crucial functions tant CAZymes, ultimately enhancing fibrolytic capac - involved in fibre break down. While taxonomically iden - ity . Co-cultivation of the bulbous anaerobic fungi tifying bacteria and fungi from complex microbial com- Caecomyces churrovis and the methanogen Methano- munities has made significant headway in recent years, bacterium bryantii resulted in a stable culture which was there are still several drawbacks that limit its usability then evaluated metabolically and transcriptionally . in fully unravelling the complexity of ecosystems like Genome analysis found that almost 1% of all adenines the equine gut. Kauter et al.  provided an overview were methylated, with at least 6% of the methylated genes of current taxonomic methods used for analysing this Wunderlich et al. Animal Microbiome (2023) 5:3 Page 12 of 17 microenvironment, and further evaluated the sequencing grow on these media and utilise the given substrates was technologies available. Taxonomically, some of the major determined by evaluating different degradation pheno - issues hindering identification of this community include, types depending on the substrate being utilized including database limitations , substantial size polymorphism change of media colour, oxidization, fungal growth, and for anaerobic fungal DNA barcodes (up to 13% polymor- halo clearing. The study demonstrated significant func - phism between clones of a single Piromyces strain ) tional diversity within taxonomic ranks, with all families and low intra-species taxonomic resolution when using having at least one strain with high degradation abilities. short read sequencing . Identifying important func- Such findings highlight the importance of identifying tions and their corresponding genes within a mixed com- fungal isolates to low taxonomic ranks (genera and spe- munity has the potential to improve our understanding cies level) to fully evaluate the role of the taxa present. of the break down of complex polysaccharides in plant A focus on enzymology rather than taxonomy may also material within the equine gut and the specific genes allow the elucidation of rare taxa involved in fibre break involved in these processes. down through production of prominent CAZymes In recent years, shotgun metagenomics has provided involved in fibre degradation. a wealth of insight into the functionality of complex Explorative microarrays, such as the ‘FibroChip’, are microbial communities, highlighting the varying func- emerging molecular tools that utilise transcriptomics tional roles of different taxa and how they work together to detect genes and their genetic variants from mixed independently and synergistically . Peng et al. , community samples . Such tools can rapidly and recently profiled the bacterial and fungal communities relatively inexpensively monitor in parallel the functional from goat faeces, targeting the taxa and enzymes respon- potential of a high number of samples, with current sible for lignocellulose break down. Briefly, faecal pellets analyses proving easier and quicker than other metatran- were cultured anaerobically on a range of enrichment scriptomics and lengthy gene annotation studies . substrate media (alfalfa stems, canary grass, xylan or sug- The FibroChip microarray was composed of thousands of arcane bagasse) with varying antibiotic treatments to bias probes targeting hundreds of CAZyme genes from eight the growth of anaerobic fungi alone, or anaerobic fungi families that were believed to have important and com- and methanogenic archaea. Shotgun metagenomics on plementary roles in cellulose and hemicellulose degrada- samples yielded 18 eukaryotic derived MAGs, the anno- tion [GH5, 9, 10, 11, 43 and 48 and CE1 and 6]. The study tated CAZymes which were categorized into either cel- was ultimately able to validate the ability of the FibroChip lulose,- hemicellulose-, or pectin/ ester-degrading. The to detect differential gene expression in bacteria and was total number of lignocellulose-active genes from eukary- even able to identify differential gene expression between otic derived MAGs was significantly higher than that of clone isolates cultivated on different substrates. The prokaryotic derived MAGs, with a number being found FibroChip was further used to analyse total RNA isolated only in fungi, indicating the incredible untapped poten- from the rumen fluid of a cannulated cow, and while tial of anaerobic fungal enzymes . Through co-cul - there were gaps in the enzymology of fungi, ultimately turing microbial groups on different media and analysing proved a valuable stepping stone towards simultaneously metabolic outputs, the study was also able to assess the taxonomically and functionally analysing complex fibro - functional performance of different microbial consortia lytic communities . in degrading lignocellulose. In line with previous studies, With the evolving use of long read sequencing plat- it was found that the fungi- and archaea-dominated com- forms such as Nanopore Sequencing from Oxford munity not only degraded several substrates better than Nanopore Technologies and single-molecule-real-time bacteria-dominated consortia, but also had significantly sequencing (SMRT) from Pacific Biosciences, the speed higher levels of cellulosomal CAZymes compared to the and accuracy at which we can screen genetic material bacterial consortia. from microbial communities for enzymatic genes and Large scale phenotypic assays are another valuable taxonomic barcodes is continuing to improve [179, method in uncovering key taxa involved in important 180]. At the moment, both technologies can rapidly targeted functions, such as fibre degradation. Recently, generate millions of full-length reference sequences 1031 fungal strains acquired from a diversity of habitats (over 10 kb) within a couple of hours , however were used in a large-scale multi-phenotyping assay to come at the cost of increased error rates compared to determine their ability to degrade non-natural industrial more traditional sequencing platforms . SMRT compounds such as plastics and dyes . This assay sequencing has been used to build fungal genomes for involved culturing each strain on six different media with functional annotation, unveiling novel fungal genera different degradation-resistant compounds, to determine from the equine gut  and elucidating the complexi- their usability as industrial biocatalysts. Their ability to ties of the cellulosomal complex from P. finnis . The W underlich et al. Animal Microbiome (2023) 5:3 Page 13 of 17 References application of these technologies in large scale enzyme 1. 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Large-scale phenotyping of 1,000 fungal strains for the
Animal Microbiome – Springer Journals
Published: Jan 12, 2023
Keywords: Equine; Microbiome; Gastrointestinal tract; Fibre; CAZyme; Anaerobic fungi; Health
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