Unravelling methanogenesis in ruminants, horses and kangaroos: the links between gut anatomy, microbial biofilms and host immunity

Additional keywords: acetogenesis, blind sacs, haustra, innate lymphoid cells, mucin.

Strategies to inhibit enteric methane (CH₄) production in herbivorous animals have been targeted as major areas for research funding because of the perceived need to curb the release of greenhouse gases into the atmosphere (Hristov et al. 2013). Because of their importance as major sources of food and fibre production for humans, ruminants have been studied more closely than other herbivores, especially in relation to aspects of their gut microbiology, gut physiology and gaseous emissions (Annison and Bryden 1998).

All animals host a large and complex biomass of microbes in their gastrointestinal tract. The microbiota present include viruses, Archaea, bacteria, protozoa and fungi that have evolved in communities that exhibit intimate between-species and species–host tissue associations. Considerable advances have been made in our understanding of the ecology of the gut microbiota of mammals and mutualistic interactions among the microbiota with the host’s immune systems have been recognised and explored (for recent reviews, see Eberl 2010; Bevins and Salzman 2011; Sansonetti 2011; Schluter and Foster 2012; Bang et al. 2014; Dishaw et al. 2014). These associations control many aspects of the physiology and biochemistry of the host, to such an extent that they are often regarded as an integral component of what has been dubbed the ‘super organisms’ (Eberl 2010). Mutualism is a two-way process and host secretions may be responsible for differences in the fermentation processes and, therefore, the end products of feed and fibre digestion in different animal species.

When ruminants digest forage diets, they produce three to four times more CH₄ (a potent greenhouse gas) per unit of digested fibre (digestible neutral detergent fibre) than do macropods (von Engelhardt et al. 1978; Madsen and Bertelsen 2012) or equids (92 ± 15 versus 28 ± 9 L/kg; Franz et al. 2010). Methane emissions represent between 6.7 ± 1.7% of the gross energy and 12.3 ± 3.1% of the digestible energy of feed ingested by ruminants, whereas these emissions represent, respectively, ~1.5 ± 0.2 of gross energy and 3.2 ± 0.7% of digestible energy for equids (Franz et al. 2010), and similarly small amounts for ‘macropods’ (Kempton et al. 1976; Madsen and Bertelsen 2012).

The among-species variability in CH₄ emissions in herbivores has been attributed to various factors. It is assumed there is an evolutionary connection between the level of CH₄ production per unit of digested feed and the site of CH₄ production in the gut. The differences in the anatomy of the fermentation organs and their position in the gut also appear to be of major significance.

The digestive tract of horses is different from that of macropods and ruminants as feed first enters the acidic stomach and then the small intestine, before being subjected to the fermentative digestion in the hind-gut (caecum–colon). Digestion of feed by host enzymes in the small intestine would not be associated with the production of gases. Therefore, on the same diet, equids will have a lower production of CH₄ per unit of dry matter digested. However, these differences can be calculated to be almost negligible with animals fed roughage-based diets without added concentrates. For instance when fed a poor-quality hay, sheep produced three-fold more CH₄ per unit of digested fibre (digestible neutral detergent fibre) than did ponies (Franz et al. 2010).

The need to explain the among-species differences in fermentation processes in the gut led to the concept for the present review that focusses on the microbial ecology of the gut of ruminants and macropods (both foregut fermenters) and horses (hind-gut fermenters). It appears that the genomes (and the resulting immune systems) of macropods and equids determine the ecology of the microbiota in their forestomach and caecum–colon respectively (Hackstein and van Alen 2010; Bevins and Salzman 2011). However, the precise nature of the physiological interactions between the host and its gut microorganisms is still not fully understood.

The intention when compiling the review was to generate and test hypotheses on the basis of currently available knowledge, but also to point to areas in which knowledge is lacking, so as to stimulate further research on the gut microbial ecology of mammals in general, and herbivores in particular. The hypotheses advanced rely on recent developments in our knowledge of the factors controlling the microbial ecology of the gut, in particular, the host’s involvement in determining the fermentative end products (gases in particular) produced in the forestomach of ruminants and macropods, and the hind-gut of horses. Consideration is focussed on the biotic mechanisms that have evolved to enable an efficient and sufficiently rapid rate of solubilisation and breakdown of complex structural components of plants (in particular lignin-associated polysaccharides) to enable herbivores to survive and grow (Wang and Chen 2009), while, at the same time, restricting gas production from fermentation in digestive organs where this could be damaging.

To establish the background information leading to these hypotheses, the opening sections of the review emphasise the major anatomical differences among the fermentative regions of the gut of ruminants, those of macropods and those of horses because these affect the microbial species composition in their gut biofilms. The importance of the abundance of different microbial species in these biofilms is emphasised, particularly of those involved with the disposal of H₂ into either CH₄ or acetate. With this background, the principal hypothesis is advanced to explain why H₂ from fermentation is necessarily directed into acetate rather than CH₄ in tubiform regions of the macropod forestomach and the hind-gut of most mammals; the reasoning, which in accord with the danger theory of Matzinger (2012), is that these fermentative organs lack physical mechanisms to remove gases that might otherwise accumulate and damage the host’s gut integrity, so the host uses its immune system to alter its gut microbiota, so as to minimise gas production. Current evidence for a two-way interaction between host immune factors and the abundance of different microbial species in biofilms involved in feed digestion, and, therefore, the acetogenic or methanogenic nature of the fermentation, is then presented.

The differences in gut anatomy between two foregut fermenters, the ruminant and the macropod, are shown in Fig. 1. Similarities between the foregut of macropods and the caecum of horses are illustrated in Fig. 2.

Fig. 1.  Anatomy of the gut of the ruminant (left) and of the macropod marsupial (right) (source: Hume 2002).

Fig. 2.  Similarities in the anatomy of the foregut of macropod marsupials and of the caecum–colon of equids (source: Hume 2002).

A ruminant uses its mouth and tongue to harvest forages during grazing or to ingest feeds offered under more intensive conditions. During the day, grazing animals typically spend more than one-third of their time foraging and at least one-third of their time ruminating. The roof of the ruminant mouth is a hard palate without incisors. The lower-jaw incisors are wide with a shovel-shaped crown and work against this hard dental pad. Premolars and molars match between upper and lower jaws. These teeth crush and grind plant material during initial chewing and also during rumination. In the mouth, forage mixes with saliva and chewing forms it into a bolus. Muscle contractions and pressure differences move the bolus down the oesophagus and on into the reticulo-rumen (the main foregut compartments, hereinafter referred to as the ‘rumen’), where contractions of the gut wall mix the contents. Newly ingested feed materials and larger particles that are less dense than the other rumen digesta often rise to form a mat of fibrous materials in close association with a gas cap on the surface of the rumen contents.

The bi-directional function of the oesophagus of ruminants allows them to regurgitate boluses mainly derived from the larger feed particles in the rumen, for further chewing and particle-size reduction. The re-chewed materials are then swallowed again with new feed added; eventually, the materials return to the rumen where the highest proportion of feed is solubilised, with the production of fermentative end products (see video, Gookin et al. 2011).

It has been proposed that, during rumination, partially digested forage is returned to the mouth where biofilm fragments that are abraded during chewing, innoculate newly gathered feed (Leng 2011); microbial fermentation in the rumen is efficient and mainly confined to the matrices of biofilm-encased microbes associated with the feed particles (Leng 2014, and references therein) and it can be likened to a continuous-flow, stirred-tank reactor (CFSTR; Hume 2002).

Macropod dentition is distinctively different from that of ruminants. The larger species of macropods have complex, high-crowned teeth. The four permanent molars on each side of both jaws erupt in sequence from front to back and move forward in the jaw with age, eventually being pushed out at the front. The molars possess cross-cutting ridges, so that tough grass or herbs are sheared between opposing teeth. The molars of smaller macropods are much simpler, so their mechanisms for grinding of feed materials into smaller units are inferior to those of ruminants. In addition, they have no mechanism similar to rumination. Nevertheless, regurgitation of food caused by violent contractions of the sacculated forestomach can occur occasionally during the day. The process has been termed ‘mycerism’ by Baker et al. (1963). The macropod eats more slowly and chews feed into much finer particles (Hume 2002). This strategy may increase the rate of feed colonisation by biofilms and enhance fermentative digestion (Langer 1984).

The wall of the forestomach of macropods is organised into three longitudinal bands of muscle, termed taeniae. Folds between the taeniae form the haustra. Contractions of the haustra propel feed boluses distally, while, at the same time, the folds selectively retain particles and release fluid. The overall effect is to squeeze fluid through the ingested feed particles, so that the fluid passes through the digestive tract more quickly than do the solids (Dellow 1982; Hume 2002; Figs 1, 2).

Haustral churning is the sequential movement of gut contents from one haustrum to the next; one haustrum expands as materials fill it, causing the muscles to contract, and the contents are pushed to the next haustrum. In this manner, remnant digesta is moved through these tubiform organs, with minimal mixing among batches. It is proposed that solid digesta is solubilised by the concerted effects of the syntrophic microbes in biofilms attached to the particles. The mechanisms may be the same wherever haustra are present in the gut compartment, including in the forestomach of the macropod and the caecum–colon of the horse and other mammals.

As the digesta move distally, particles decrease in size as a result of microbial fermentation. Radiographic studies indicate that ingested food is initially retained in the cranial region (Dellow 1979; Langer et al. 1980; Richardson 1980) and, although localised mixing of a single dose of a marker is effective, the marker does not mix with the entire contents of the forestomach. Digesta markers were observed radiologically to be transported slowly along the length of the forestomach, so that, in the frequently fed animal, after a period of 6 h, newly ingested food was not marked with previously administered contrast medium (Dellow 1979). These observations are evidence of bolus or tubular flow, perhaps commencing in the region of the second haustral pouch, as it appears that markers are mixed in sacculated forestomach and the first tubular components.

The finding of an accelerated solute marker excretion from the forestomach of macropods (Dellow 1979) is highly suggestive of a propulsive (aborad) peristalsis of the forestomach haustra. The forestomach of the macropods appears, therefore, to be analogous to a series of CFSTRs preceded by a continuously stirred reactor (Hume 2002), which is the sacciform component at the junction of the oesophagus and the tubiform stomach. The sacculated stomach has two small blind sacs, viz. the parietal sac and medial blind sac (Langer et al. 1980; Shoeib et al. 2015). The structure of the macropod’s foregut resembles that of the caecum in the horse (see Fig. 2). In the discussion that follows, it is speculated that the parietal blind sac of the macropod forestomach has a function similar to that ascribed by Bollinger et al. (2007) to the human appendix. It is a source of mucosal protobio-films containing immune agents (discussed in more detail below) that, having developed on the gut mucosal surface, adhere to and inoculate incoming feed materials with appropriate species of microorganisms.

Hume (1999) suggested that, on the basis of chemical-reactor theory, digesta mixing and movement in the macropod forestomach is best understood by viewing the forestomach as a series of CFSTRs, whereas the foregut of the ruminant is better documented as a single CFSTR. The CFSTR model, which features continuous flow of material into and out of a spherical reaction vessel, certainly describes the rumen well. In an ideal CFSTR, mixing is continuous and reactant concentrations are uniform throughout the vessel and, at steady-state, inflow rate equals outflow rate.

The presence of a sequence of CFSTRs in the macropod foregut suggests that very different digesta flow patterns must occur in their fermentative compartments (as envisaged in Fig. 3), from those in the forestomach of ruminants, which have been well documented (Fig. 4).

Fig. 3.  Envisaged mixing compartments and digesta flow in the macropod marsupial forestomach, which is composed of a sacculated section followed by haustrated tubiform section that resembles a series of continuous-flow, stirred tank reactors; there is minimal retrograde digesta flow between haustral pouches, before residual solids enter the acid hind-gut.

Fig. 4.  Movement of digesta within the ruminant forestomach. This chamber resembles a single spherical stirred-tank reactor. E, oesophagus; R reticulum; Ro reticulo-omasal orifice; C, cranial sac; D, dorsal rumen; V, ventral rumen; DB, dorsal blind sac; VB, ventral blind sac; O, omasum; and A, abomasum.

The sacciform morphology of the rumen, and the separation of small particles and return of large particles during rumination, increases the retention time of feed particles and improves the digestibility of structural plant cell-wall materials. Vigorous mixing, together with the rumination cycle (Gookin et al. 2011), causes feed particles to fragment, increasing the surface area available to microbial colonisation, and allows biofilm particles abraded from other feed particles to attach to newly ingested feed materials. This, in turn, reinforces the establishment of biofilms on newly arriving feed material.

Herbivores obtain the majority of their energy needs by digesting simple and complex polysaccharides, proteins and fats in feed materials. The organs in which fermentation occurs retain large amounts of cellulosic biomass for extended periods, enabling the feed organic matter (OM) to be more extensively solubilised and then fermented to volatile fatty acids (VFAs) that are the main source of absorbed energy substrates for the host (Hungate 1966). In foregut fermenters, most fermentation occurs before digesta reaches the host’s gastric stomach and small intestine and there is only subsidiary OM fermentation in the caecum and colon. In hindgut fermenters, digesta passes through the gastric stomach and small intestine, enabling sugars, fats and amino acids to be absorbed, before entering an enlarged fermentative region of the hindgut (caecum and colon) where the majority of the fermentative microbes are present. In forestomach fermenters, the microorganisms formed during fermentation are rich sources of protein (amino acids) for the host after they move into the lower gut and are digested. However, in hindgut fermenters, microbial proteins are lost to the animal when they are voided in faeces.

In ruminants and macropods, fermentation end products provide the major source of energy to the host in the form of VFA and microbial proteins are the major source of essential amino acids. Anaerobic microorganisms digest the majority of the feed OM via the Embden–Meyerhof–Parnas pathway of metabolism to pyruvate, and then to acetyl CoA and VFA (Hungate 1966). For this pathway to be efficient, there are the following two main requirements:

1. The adherent microbes must be closely associated in syntrophic associations within biofilms to facilitate efficient digestion of fibrous materials by hydrolysing their complex carbohydrates to soluble sugars and then to VFAs (Stoodley et al. 2002; De Mulder et al. 2016).

2. Glycolysis and oxidative decarboxylation of pyruvate result in the release of NADH⁺ +H⁺, which must then be oxidised to NAD⁺ with the release of metabolic hydrogen, [H], to permit fermentation to be continuous. Metabolic [H] is released as gaseous H₂ or removed by methanogenesis or acetogenesis.

If the [H] is converted to gaseous H₂, large volumes of gas are produced (although the volume is lower by a factor of 4 if [H] is oxidised to CH₄). The solubilities of both H₂ and CH₄ are low, so these gases must be removed quickly to prevent gut distension, e.g. by eructation or as flatus. If methanogenesis is suppressed, the [H] must be removed in other reactions, usually by acetogenesis, to avoid possible damage to the gut (discussed below). Importantly, no gas is produced when acetogenesis is the [H] sink.

An example of the amounts of H₂ or CH₄ that can be produced (in a stable rumen at pH 7 where CO₂ is mostly present as bicarbonate and therefore not problematic) follows. A steer fed a high-roughage, low concentrate-based diet, weighing 320 kg liveweight and growing at 1 kg/day produces ~135 g or 8.43 mol of CH₄ daily. In gaseous form, this represents 189 L of CH₄. If the gas produced had been H₂, the volume of gas excreted would have been 756 L. If either of these gases were released into a rumen capable of holding ~30 L of liquid, in the absence of a release mechanism (the eructation reflex), the pressure generated would have distended the rumen and caused damage to its epithelium, as well as potentially also damaging other vital organs. Accordingly, the ruminant forestomach has evolved with anatomical and physiological adaptations to prevent gaseous distension.

Gas that accumulates in the apical part of the rumen can be quickly eliminated by eructation (Gookin et al. 2011) and eructation seems to be effective for even high rates of H₂ production, e.g. when CH₄ production is inhibited by selective toxins (Mitsumori et al. 2012). Nevertheless, when the eructation mechanism is inhibited by the formation of foam, gases build up and massive distension (bloat) of the organ can result (Clarke and Reid 1974). This is a rare phenomenon, mostly restricted to cattle on starch-rich feeds or on legume-based pastures subject to certain plant growth conditions; these situations would have been unlikely to occur during evolution and, so, protective mechanisms have not developed.

Where there is no gas-escape mechanism, the build-up of gas could damage cells by impairing NAD⁺ regeneration and inhibiting respiration; it could also disrupt the gut mucosa and, when extreme, gas production can lead to pneumatosis intestinalis (Gibson et al. 1993). This condition is characterised by gas cysts in the bowel wall. Excessive gas pressure is, therefore, a major threat to the integrity of the gut. The onset of physical damage to the gut mucosa may provoke an immune reaction according to the danger theory of Matzinger (1994, 2012). This theory is an extension of the conventional understanding of the immune system that allows the host to differentiate ‘self’ from ‘non-self’; Matzinger and colleagues have argued that the immune system not only reacts to foreign substances but also responds to alleviate situations that are potentially ‘dangerous’ to the animal. Pradeu and Cooper (2012) preferred ‘damaging’ as a more appropriate description.

Although macropods do have a form of eructation, this occurs only a few times per day and so is not a gas-release mechanism. Even so, it may allow the microbial mix in the forestomach to be inoculated with microbes from the more distal haustra. Similar retrograde movements of gut contents occur in the rabbit where reverse (orad) peristalsis of the colonic haustra separates very fine particles, including bacteria, from other digesta and concentrates these in the caecum (Ehrlein et al. 1983).

In the rumen, most metabolic [H] is removed by CH₄ generation. If methanogenic activity is suppressed, metabolic [H] has to be oxidised in other ways to enable fermentation of OM to continue. This is achieved mainly by acetogenic microbes that use [H] to reduce CO₂ to acetate and have a higher H₂ threshold than do methanogens.

The H₂ threshold is the minimum H₂ concentration in an anaerobic microbial ecosystem during the reduction of only one specific terminal electron acceptor (Lovley 1985; Cord-Ruwisch et al. 1988; Lovley and Godwin 1988; Conrad 1996). For methanogenesis, the H₂ threshold in parts per million by volume (ppmv) is 6–120 (Conrad et al. 1983; and, for acetogenesis it is 430–4660. In mixed populations, homo-acetogens are, therefore, disadvantaged and often prevented from establishing in biofilms on feed particles. Owing to the presence of multiple hydrogenases with distinct properties, production and uptake of H₂ can occur simultaneously within a single bacterial species or between microbial colonies within the biofilm (Fauque et al. 1988). The release of H₂ in the biofilm matrix can be discontinuous and the steady-state partial pressure of H₂ is potentially maintained by the ability of some acetogens to increase acetate production at high H₂ pressures and to produce H₂ from acetate whenever the partial pressure falls below a certain level. The acetyl-CoA or Wood–Ljungdahl pathway is the major pathway of acetogenesis and appears to be totally reversible (Ragsdale and Pierce 2008). Acetogens are metabolically versatile and almost all known acetogens can utilise alternative terminal electron acceptors such as nitrate. Some acetogens utilise the acetyl-CoA pathway to grow autotrophically on H₂ and CO₂; others grow heterotrophically or mixotrophically on a variety of organic compounds (Ragsdale and Pierce 2008).

The ability of homoacetogens to grow mixotrophically by metabolising sugars to generate ATP in the early stages of development of the biofilm would allow a larger pool of acetogens to develop so that, when the availability of sugars declines as the digesta remnants move through the haustra, the acetogens could revert to using H₂ as their main energy source. The higher partial pressure of H₂ needed to support homo-acetogenesis as against that required by methanogens could be maintained in biofilms by the ability of acetogens to reverse the acetogenesis reactions as digesta move distally in the macropod forestomach. In contrast, methanogens and reductive acetogens grow at close to their thermodynamic limits and close to the minimum quantum of free energy needed to sustain microbial growth (Schink 1997; McInerney et al. 2009).

The majority of microbes in nature, including those that ferment carbohydrates in the gut of mammals, exist in biofilms attached to solid surfaces (see Box 1).

The critical role of biofilms in the fermentation of complex OM in the rumen has been reviewed by Craig et al. (1987), McAllister et al. (1994), Cheng et al. (1995), McAllister and Cheng (1996), Mayorga et al. (2007), Edwards et al. (2008), Leng (2014) and de Mulder et al. (2016). Many reviewers have emphasised the point that microbes must be attached to, or closely associated with feed particles, so as to enable efficient degradation of feed materials to occur. However, the concept that attached rumen microorganisms are organised in complex biofilms that facilitate mutualistic associations among co-located microbial species that also facilitate feed degradation has only recently been highlighted (Leng 2014; De Mulder et al. 2016, and references therein; summarised in Box 2).

Much more is known about the biofilms in the gut of ruminants than of those in the guts of the other herbivores. The suggestion that biofilms promote a sufficiently high rate of solubilisation of fibrous feeds to meet the protein and energy requirements of ruminants (Weimer et al. 2009) supports the concept that dense biofilms must be responsible for the efficient digestive activity in the forestomach of macropods and the caecum–colon of equids. Pope et al. (2011) indicated that muramidase found in the foregut microbiome of the Tammar wallaby can direct cell aggregation and biofilm formation and this provides some indication that fermentative digestion is undertaken mainly by biofilm microbiota. However, in omnivorous or carnivorous mammals, a major ecological niche for gut microbiota is in biofilms attached to gut-wall mucosal cells (Sommer and Bäckhed 2013).

Useful information about biofilm-like structures can be obtained from many sources. Most aquatic ecosystems contain planktonic transparent exopolymer particles (Bar-Zeev et al. 2012). These organic microgels are highly surface-active (Bar-Zeev et al. 2012) and, when they contain microbial colonies, they are termed proto-biofilms. The transparent exopolymer particles in seawater originate from dissolved polymeric OM or from preformed biofilms and have most of the characteristics of ‘fledgling’ biofilms; they attach to surfaces and provide habitat for planktonic microbes to become sessile and proceed to develop colonies and to self-produce a matrix of hydrated extracellular polymeric substances that form their immediate environment. In the rumen, similar exopolymer particles seem to originate as sloughed biofilm fragments released from the surface of feed particles during rumination (Leng 2011, 2014), which is in accord with observations that 80–90% of the microbes in the rumen appear to be associated with particulate matter (Rodríguez et al. 2003). In addition, 80% of the microbes leaving the rumen have been particle-associated (Krebs 1987), indicating that, before leaving the rumen, a high percentage of bacteria and Archaea have moved between feed particles, without apparently mixing in the planktonic microbial pool (Krebs 1987). However, this does not preclude the likelihood that planktonic bacteria can adhere to the solid feed particles as a focal point for biofilm development (Huws et al. 2013). Both processes probably occur simultaneously and are complimentary.

The above arguments suggest there are significant differences between the way biofilms develop in the rumen and in the forestomach of macropods, and the caecum–colon of mammals in general. In the human appendix, for example, biofilms initially develop in association with epithelial cells linked to lymphoid tissue (Bollinger et al. 2003, 2007) and are thought to detach from the host epithelial tissue and then attach to digesta particles on which they mature as they move distally through the caecum (Bollinger et al. 2007). Accordingly, it appears probable that mucosal biofilms develop in the blind sacs, proximal to tubiform areas of the mammalian gut, where fermentative digestion of feed particles and other solids occurs. On this basis, it is likely that these proto-biofilms could develop in all blind sac structures containing lymphatic tissues. In macropods, the site of proto-biofilm development would be cranial to the forestomach and, in equids and ruminants, before the caecum.

In support of the above discussion, Bollinger et al. (2003, 2007) showed in humans that, following the collapse of the caecal microbiota from antibiotic treatment, mucosa-produced biofilms that had apparently been protected in the appendix were able to re-inoculate the caecum with normal microbiota. On this basis, the blind sac has been termed a ‘safe house’.

In summary, a crucial part of the hypothesis advanced later in this essay to explain the observed differences in fermentation characteristics and gas production between herbivores is that microgels containing immune factors formed in the parietal blind sac of the foregut of macropods, and in the blind sacs of the caecum of horses and ruminants, may inoculate proto-biofilms before they attach to particles and develop into mature biofilms as digesta moves through the tubiform forestomach and colon. These rudimentary biofilms, which form in close association with a host’s mucosal epithelial cells, would have the same function as proto-biofilms that can quickly mature to form ‘hotspots ‘of biofilm microbes on solid particles (Bar-Zeev et al. 2012). In the rumen, no such mechanisms are necessary and are not present.

The hypothesis advanced herein involves a dynamic interplay between the animal and its gut microbiota; this interplay has been a focus of considerable recent research, particularly where the colonising bacteria play important roles in the digestion of food/feed and play a major role in the health of animals (reviewed by Bevins and Salzman (2011), Schluter and Foster (2012) and Dishaw et al. (2014) and summarised in Box 3).

The term, microbiome, has been coined to describe all of the genes in the many different species found in the gut microflora. The abundance of individual species in the gut can alter markedly, in a matter of days, such as, for example, in response to changes in diet and the kinds of genes expressed (Ha et al. 2014). Currently, researchers are trying to understand the links between the dynamic activities of microbial communities and host biology and pathobiology (e.g. Brugman and Nieuwenhuis 2010). Even though the composition of these mutualistic communities of the digestive tract is variable, in the human at least, it appears that the overall microbiome is established and actually entrenched during birth, or shortly thereafter. This is apparently also so for the macropods (Peel et al. 2013) that give birth to partially developed fetuses (with a well developed head and front legs but an under-developed gut); these fetuses climb from the vagina into the pouch and attach to a teat at a very early stage of development (Tyndale-Biscoe 2005). During this period, a gut microbiome may partially establish before the animal is fully exposed to the external environment.

Molecular analysis based on bacterial 16S rRNA sequences has revealed that more than 1000 different species of bacteria are represented in the human gut microbiome (Eckburg et al. 2005) and most of these species have not been able to be cultured in vitro. Dominant bacteria are being identified in humans by using these new tools that are now also being applied to the rumen (Bath et al. 2013; de Mulder et al. 2016) and the macropod forestomach (Godwin et al. 2014). Recent molecular analyses of the composition of rumen ecosystems have demonstrated that there is great complexity within the myriad of species present in biofilms in any one location (de Mulder et al. 2016). The factors that are involved in the establishment of a specific microbiome in the gut, and those that influence the microbiota at the various anatomical sites in the gut are major areas for research (Ley et al. 2008).

The biofilm mode of fermentation in the macropod forestomach has received little study but, as argued by Wang and Chen (2009) and Weimer et al. (2009) for ruminants, it would not be possible for macropods to digest plant OM at a sufficient rate to meet nutrient requirements without syntrophic growth of microbes in close association with feed particles. In macropods, biofilms probably develop on the mucosal lining of the parietal blind sac or possibly on the apical structures of the sacculated forestomach. The medial blind sac is not likely to be a site of biofilm development, as its epithelium consists of non-glandular mucosa (Shoeib et al. 2015).

In the present essay, it is speculated that, when the proto-biofilms detach from the mucosa, they contain host immune secretions that may include antimicrobial peptides (AMPs), immunoglobulins (IGs), innate lymphoid cells (ILCs) and mucin (Dishaw et al. 2014). The presence of these secretions either prevents the establishment of methanogens or kills them, creating a niche for acetogens to become the major hydrogenotrophs present in the biofilm community. The immune cells responsible for these secretions appear to recognise ‘non-self’-molecules by numerous membrane-bound, cytosolic or secreted receptors (pattern-recognition receptors) and are the first effectors of the population balance of Archaea, bacteria, fungi or viruses (Banchereau and Steinman 1998). By recognition and binding of microbe-associated molecular patterns on the surface of microorganisms, the innate immune responses are activated so that there is the release of AMPs or production of cytokines or activation of the complement cascade (Hart and McKenzie 1990; Banchereau and Steinman 1998; Lipscomb and Masten 2002; Bang et al. 2014).

The potential for hydrogenotrophic organisms in biofilms to be recognised by the immune system will be related to their specific chemical structures, or to other factors such as disturbances to the mucous layer in the intestines. The absence of methanogenic Archaea would be likely to allow the level of H₂ to increase in the biofilm matrix and the ubiquitous acetogenic population to grow and expand, removing excess H₂ and maintaining its partial pressure at levels favouring acetogenesis without compromising the rate of fermentative digestion. If this is true, then blind sacs are more than just ‘safe houses’ that inoculate the gut digesta following a disturbance in the ecosystem The biofilms from the blind sac continuously control the nature of the microbial associations in biofilms on solids in the tubular fermentative area of the gut and limit CH₄ production at sites where gas is not easily expelled. This is in accord with Smith et al. (2009) who suggested that the driving force for the evolution of biofilm development in the blind sac of the proximal large bowel of vertebrates provided a means of maintenance of their normal bacterial populations.

In macropods, the development, subsequent growth and maturation of the mucosal-derived biofilm may be similar to that of biofilms in rumen digesta, except that acetogens are more numerous than methanogens in their layered structures. It is suggested that the biofilm communities on particulate digesta develop and mature as they move through the haustrated gut, progressively losing their immune agents as these are hydrolysed by proteolytic bacteria (Ganz 2003). In the terminal part of the macropod forestomach, secretion of acid prevents any local intrusion of methanogens. However, in the distal colon, there is greater potential for methanogens to develop.

The hypotheses proposed to explain these differences are outlined in Box 4.

The fermentation process in the haustrated forestomach of macropods and the hind-gut of the horse differs from that in the rumen because, as discussed already, batches of digesta move as individual boluses towards the lower gut. At any particular distance from the sacculated forestomach in the tubiform organ (or haustral pouch), biofilm consortia can be expected to be at the same stage of development, growth, maturation and detachment. This expectation is reinforced by the fact that differently sized particles travel at the same rate during digesta passage through the forestomach of macropods (Munn et al. 2012).

Biofilms with a high microbial digestive ability are always composed of complex multi-species layers of microbes or associated aggregates of each species (Stoodley et al. 2002). In the absence of information to the contrary, acetogens in the forestomach of macropods are assumed to be associated with the biofilms in or on feed particles and probably also attached to the epithelial wall of the forestomach. In humans, biofilms on gut mucosal surfaces adhere to the mucosa, with cell-to-cell adhesion leading to multi-structure formation. These biofilms consist of colonies of microbes that are embedded in a matrix of self-produced extracellular polymeric substances that mediate protective functions and nutrient supply and, most importantly, facilitate H₂ transfer (Bang et al. 2014).

Acetogens would need to be distributed in both the outer layers of these biofilms (to remove H₂) and close to sites of attachment on the solid digesta where substrates such as sugars are produced by enzymatic hydrolysis of the structural carbohydrates. In the rumen, the biofilms present on feed particles would be at varying stages of maturation. This is because, as already noted, the rumen is essentially a continuous fermentative reactor where digesta from sequential meals are well mixed.

In the rumen, the dominant means of removal of H₂ is by methanogenesis, even when ruminal microbes capable of reductive acetogenesis are present (Morvan et al. 1994; Pinder and Patterson 2012). In contrast, reductive acetogenesis appears to dominate as the major mechanism for H₂ removal in the macropod forestomach (Kempton et al. 1976; Madsen and Bertelsen 2012; Godwin et al. 2014) and methanotrophic Archaea are virtually absent (Ouwerkerk et al. 2009; Klieve et al. 2012).

In anaerobic regions of the gut, and in the absence of dietary nitrate or sulfate, it appears that acetogenesis can become significant only if CH₄ and formate syntheses by methanogens are inhibited by AMPs and the partial pressure of H₂ is elevated. The latter may be maintained at the threshold pressure needed to support acetogenesis by switching the direction of the Wood–Ljungdahl pathway (Hattori et al. 2005; Zinder and Koch 1984). This pathway is found in all known acetogens and methanogens and is used in both the oxidative and reductive directions (Ragsdale and Pierce 2008). These organisms apparently still generate ATP when the pathway is reversed, but how they do this is unknown (Zinder and Koch 1984; Hoehler et al. 2002). Leng and Leonard (1965) found that when labelled acetate (methyl carbon labelled with tritium (³H) and carboxyl carbon with ¹⁴C) was infused into the rumen of sheep, the ³H : ¹⁴C ratio in rumen fluid acetate declined substantially over time, indicating that the methyl tritium was being replaced with unlabelled H. The labile nature of the methyl group of acetate is evidence that microbes that can utilise the Wood–Ljungdahl pathway are present in the rumen and may play an important role in controlling the H₂ concentrations in the biofilms.

Acetogens appear to be present in the digestive tracts of all mammals. However, they appear to be competitive with the hydrogenotrophic Archaea only in certain sites where acetate is the electron sink. In environmental biofilms, sulfate- or nitrate-reducing bacteria are able to lower H₂ partial pressure (Cord-Ruwisch et al. 1988) and these bacteria partially or totally out-compete reductive acetogens. Under most feeding conditions, sulfate or nitrate are present in only small amounts in the diet but, when concentrations of sulfate or nitrate are elevated by direct dietary supplementation, these salts become the dominant H₂ sinks ahead of CO₂ (Lee and Beauchemin 2014).

Recent research has indicated that unknown genetic factors are involved in determining the predominant hydrogenotrophic organisms in the gut of mammals (Hackstein and van Alen 2010; Dishaw et al. 2014). There appears to be a strong reciprocal and mutually beneficial interaction between the immune system and the microbial profile in the colon of humans (Bollinger et al. 2007; Dishaw et al. 2014). Because syntrophic associations of microbes in biofilms are essential for efficient and rapid digestion in ruminants, it is assumed here that biofilm formation is also essential for the efficient digestion of feed in the forestomach of macropods and hind-gut of horses. It is envisaged that these mucosa-associated biofilms contain immune agents obtained in a way similar to that described by Bollinger et al. (2007) for the human appendix. The parietal blind sac of the macropod forestomach may have a secretory function and also be the site where these proto-biofilms develop before being released into the sacculated forestomach. Once detached, these proto-biofilms may then re-attach to feed particles and continue to develop in the sacculated part of the foregut and as the digesta bolus proceeds through the haustrated forestomach. There is increasing knowledge of both host-mediated biofilms in the human colon (Bollinger et al. 2003; Bang et al. 2014) and of the role of biofilms in the rumen (see Costerton 2007; de Mulder et al. 2016); however, no studies have compared and contrasted the development of host-mediated biofilms in other mammals. It was, therefore, necessary to extrapolate from the available literature when developing the hypotheses advanced in the present essay.

Conditioning of a feed-particle surface for biofilm and microbial attachment begins immediately after the feed enters the mouth. During chewing, feed is mixed with submaxillary salivary mucin. The key characteristic of mucin is its ability to form gels and it is, therefore, a component in most gel-like secretions. The proposed mechanisms for mucosal biofilm formation take into account the recently recognised role of mucosal-associated microgel particles resembling proto-biofilm in facilitating and accelerating this process. It is proposed here that this is analogous to the inoculation of inert surfaces with biofilms in aquatic environments, as discussed by Bar-Zeev et al. (2012).

It is further argued that mucosal biofilms (as microgel particles) develop on, and are released from the uppermost mucosal layer of the parietal blind sac (see Box 4), and that these immature biofilms harbour host secretions containing mucin and possibly immune elements such as the innate lymphoid cells (ILCs) that produce antimicrobial peptides (AMPs) and immunoglobulins (IgA; Dishaw et al. 2014). The attachment of bacteria and additional microgel particles in the released microgel probably initiates the early development of feed particle-attached biofilms that have a complement of host secretions, including substrates such as mucin (which may support acetogens) and immune agents that prevent growth of methanogens (and, therefore, predetermine the microbial mix that develops early as the biofilm matures and passes through the haustrated components of the forestomach/caecum). The envisaged series of events that occurs as the biofilm develops on feed particles and moves through the forestomach of the macropod is illustrated in Fig. 5.

Fig. 5.  Visualisation of the series of events that promote acetogenesis in syntrophic fermentative digestion in the tubiform forestomach of macropods. Notes on the numbered processes: 1. Host-mediated biofilm development, immune inclusion and inoculation with microbes from luminal contents while attached to the mucous epithelium of the parietal blind sac. These microbes may attach from the planktonic populations or from proto-biofilms developed on the apical areas of the blind sac and they multiply using mucin and other epithelial cell secretions. The initiating organisms may be mainly homoacetogens as they can utilise the soluble sugars (from the feed or in mucin) and H₂ while the growth of methanogens is specifically inhibited by the presence of antimicrobial peptides (AMPs) and immunoglobulin A (IgA). The organisms continue to produce extracellular polymeric substances (EPS) in the luminal contents after detaching as proto-biofilms from the epithelium. 2. The proto-biofilms and planktonic microbes in the luminal contents adhere to the surface of newly ingested feed particles and hydrolyse the complex polysaccharides. Acetogens providing the main control of the partial pressure of H₂ as the biofilm expands into the feed particle. 3. Inoculation of feed particles is facilitated by digesta mixing in the sacculated forestomach and probably in the first haustral pouch. In the sacculated compartment of the forestomach, microbes quickly degrade the soluble components of the feed. 4. Development of the syntrophic fermentation involves both feed particles and mucosa-derived components of the biofilm as it moves distally in the tubiform section of the forestomach. The epithelial derived IgA and AMPs may become substrates for the biofilm microbiota as plant particles move into the distal haustra. 5. Maturation of the biofilm and solubilisation of the structural components of forage are optimised as the boluses move through the haustral pouches towards the acidic stomach where the microbes are killed and, in the small intestine, lysozyme and other proteolytic enzyme secretions release the amino acids for absorption.

In recent times, the complex interactions of the immune system that play a role in controlling gut microbiota have been revisited with the discovery of ILCs that have roles in mediating immune responses and regulating tissue homeostasis (Walker et al. 2013; see Box 5).

The ILC family resides predominantly in mucosal issues, particularly in the intestinal tract, where ILC3s (Box 5) have a crucial role in mediating a delicate balance between the symbiotic microbiota and the intestinal immune system (Walker et al. 2013). The LTi cells, an ILC subset, have been recently recognised as important regulators of lymphoid-tissue architecture after birth; they have been shown to mediate immunity to enteric pathogens (Sonnenberg et al. 2011) and also appear to trigger release of anti-microbial peptides (Eberl et al. (2015). The scope of the present review prevents further exploration of the possible role of ILCs in the biofilm microbiota that are proposed to develop in association with the lymphoidal tissue associated with the proximal large bowel of some herbivores or the paretial blind sac of the forestomach of macropods. However, the above discussions provide some evidence for the assumption that methanogens are controlled by immune factors in the gut of some mammals.

Goblet cells, epithelial cells, macrophages and dendritic cells are the major cellular constituents of the innate defence system, and the mucous layer that coats the epithelial lining of the gut compartments contains mucin (Corfield et al. 2000). Mucin is the key polymeric, visco-elastic and protective component of mucus. In addition to mucin, mucus contains molecules of the immune systems such as IgA and AMPs that facilitate the clearance of pathogenic organisms (Kim and Khan 2013; Hasnain et al. 2013) or, as postulated here, other non-pathogenic organisms capable of producing end products such as H₂ and CH₄ that may damage the host’s gut integrity. Communication/reaction between immunoglobulins and complexes with commensal bacteria and the epithelial cells lining the mucosal surfaces of the gut has been implicated in the symbiotic host–commensal relationship (Blais Lecours et al. 2014). Thus, mucin, as part of the endothelium-derived biofilm on digesta particles, may have an important role in shaping the microbial biome (Koropatkin et al. 2012), which appears to have been under-recognised.

Mucin is secreted as large aggregates of proteins with molecular masses of 1–10 million Da. The high content of sugars in mucin could give the biofilms considerable water-holding capacity. Mucin may also partially protect immune secretions such as AMPs and IgA from hydrolysis by resident microbes and provide a readily fermentable source of substrate to rapidly increase the rate of growth of acetogenic bacteria in the early stages of biofilm development in the tubiform regions of the gut. The mucin secreted from specialised epithelial cells exchanges Ca²⁺ for Na⁺ and expands considerably, producing a visco-elastic gel with interwoven molecules that, combined with other secretions, forms part of the mucosal biofilm (Bollinger et al. 2007; Kim and Khan 2013). Mucin secretion by the epithelial cells appears to be obligatory, providing structures for attachment of cells and host-derived chemicals for the development of biofilms on feed particles (Bollinger et al. 2007; Smith et al. 2009; Dishaw et al. 2014).

An additional speculation is that fermentation of the closely available mucin sugars within the biofilm matrix may also play a role in activating some AMPs. Schroeder et al. (2011) suggested that activation of AMPs through reduction of disulfide bonds could occur in the gut of humans, mice and probably other species (this was termed environment-dependent activation; Ostaff et al. 2013). The AMP termed human β-defensin 1 (hBD-1) is constitutively expressed by epithelia but, in comparison with other AMPs, its anti-microbial activity is low. However, Schroeder et al. (2011) found that, after reduction of the three disulfide bonds in hBD-1, its anti-microbial activity was strongly enhanced. Reduced hBD-1 is able to kill Gram-positive anaerobic bacteria of the normal gut flora of the human as well as opportunistic pathogens, whereas the oxidised peptide has not shown such activity. A possibility here is that, in the proto-biofilm stage of development, AMPs are in the oxidised form but, with movement of digesta down the tract and the commencement of fermentation, the resultant increase in the partial pressure of H₂ ensures a favourable reducing environment in the biofilm matrix that activates the AMPs that kill (lyse) any Archaea that are present.

The bacterial membrane is made up of fatty acid esters. In contrast, the lipids in Archaeal membranes are derived from glycerol di-ethers of isoprenoid alcohols arranged as a bilayer or, in some Archaea, they consist of glycerol tetraethers that form mono-lamellar membrane patches (Fig. 6).

Fig. 6.  Archaeal cell walls lack peptidoglycans that are universally found in all bacterial cell walls and instead contain pseudo-murein (or pseudo-peptidoglycan), polysaccharide or protein (De Rosa et al. 1986). Top: Membrane structure of Archaea. (1) Isoprene side chain; (2) ether linkage; (3) L-glycerol; (4) phosphate moieties. Middle: bacterial and eukaryan membrane. (5) Fatty acid; (6) ester linkgage; (7) D glycerol; (8) phosphate moieties. Bottom: lipid bilayer of bacteria, eukarya and most Archaea. (9) Lipid bilayer; (10) lipid monolayer in Archaea.

Archaeal cell walls lack peptidoglycans that are universally found in all bacterial cell walls and instead contain pseudo-murein (or pseudo-peptidoglycan), polysaccharide or protein (De Rosa et al. 1986). The Archaeal cell-wall polymers are structurally diverse, ranging from pseudomurein surface-layer (S-layer) proteins to methanochondroitin (Kandler and König 1998). These differences offer major clues to the roles of AMPs in shaping the microbial ecology of the gut (Sansonetti 2011). The AMPs have only recently been examined in relation to their ability to kill Archaea. Bang et al. (2014) clearly demonstrated the effects of different synthetic AMPs on the growth of Methanosarcina mazei, Methanobrevibacter smithii and Methanosphaera stadtmanae isolated from the human gut. Methanogens in general were highly sensitive to derivatives of human cathelicidin, porcine lysin and a synthetic anti-lipopolysaccharide peptide (Lpep). Sensitivities differed markedly among the methanogen species, but disruption of the unique cell envelope of Archaea occurred within minutes of exposure to all AMPs tested. This demonstrated that AMPs released by epithelial cells are a potent defence mechanism targeting methanogenic Archaea (Bang et al. 2014). The effects of the AMPs add support to the hypothesis that host-produced AMPs are secreted into the developing proto-biofilms while they are associated with the lining of the blind sac and, when AMPs are released to the lumen of the forestomach of macropods, they prevent methanogens from establishing, thus creating a more favourable environment for the growth of acetogens. This provides a plausible explanation for the differences in hydrogenotrophic microbial populations in the forestomach of ruminants and macropods, and the also the caecum of ruminants and hindgut fermenters (where AMP control of Archaea may be weakened as remnant digesta move towards the rectum).

The pouch of macropods is a rich source of AMPs (Peel et al. 2013). Considering that the under-developed fetus is attached to mother’s teats in the pouch for an extended time, it is hypothesised that it is exposed to early colonisation and so can potentially develop a wide range of AMPs. The recent whole-genome sequencing of the Tammar wallaby has allowed discovery and characterisation of novel AMPs in this species (Daly et al. 2008). Further studies have shown strong broad-spectrum activity of these AMPs (Wang et al. 2011), indicating their likely important role in the immune system of macropods in particular. From an evolutionary view point, if AMPs that specifically kill methanotrophic Archaea are responsible for the dominance of acetogenesis over methanogenesis in tubiform fermentative areas of the gut, this activity could have evolved to counteract the potential damage associated with gas pressure in tubiform organs.

The hypothesis advanced to explain the differences in CH₄ production in ruminants versus other herbivores is that host-determined immune secretions ensure that methanogens are prevented from establishing in the fermentative chambers that would be damaged by a build-up of gas pressures; methanogenesis is suppressed and acetogenesis is promoted and supported in biofilms associated with digesta particles in these vulnerable gut regions. The hypothesis is summarised in Box 6.

Anaerobic fungi grow within solid components of feed (Gordon and Phillips 1998) and are found in the rumen and also the forestomach of the macropod (Dellow et al. 1988). They produce sporangia that release zoospores that actively invade insoluble plant materials, particularly in the areas where damage to the waxy surface of feed has occurred. Penetration of fungal rhizoids throughout plant particles weakens the structures and promotes more rapid reduction of particle size and greater access for other organisms. The fungi are in close contact with the biofilm consortia or can be considered as an extension of the biofilm into the solid plant particles (Leng 2014). All species of phycomycetous fungi that have been isolated from the gut of animals are obligate anaerobes. Despite this, they have been isolated successfully from the saliva and faeces of ruminants, which suggests that there is an aero-tolerant stage in their life cycle (Brookman et al. 2000). The life cycle of the anaerobic fungi is easily envisaged in the rumen; however, if the sporangia are produced and release zoospores in the more distal haustra in the macropod forestomach, back flow of digesta may be needed to continually re-inoculate the sacculated area or the first haustral pouch. There is potential for this to occur, as merycism periodically returns partially digested feed to the mouth. The vigour of the muscular contraction in merycism suggests that there may be an intermittent but forceful return of digesta from the more distal haustral pouches to the sacculated forestomach, thereby inoculating it with fungal spores and also bacteria and protozoa. Alternatively, macropods may be inoculated by ingesting aero-tolerant spores on feed; however, this appears unlikely.

Protozoa are present in the sacculated forestomach of macropods (Dellow et al. 1988). They may be prevented from passing distally in the liquid gut contents by attachment to larger particles or the wall of the forestomach, being released only when soluble sugars are present in the feed. A similar cycle of attachment and release is seen with protozoa in the rumen (Williams 2007). Rumen protozoa exhibit hydrogenosome activity (Williams 2007) and methanogens associate closely with the protozoa to take advantage of the release of H₂ during fermentation. Whether protozoa in the forestomach of macropods also exhibit such hydrogenosome activity, but with acetogens replacing methanogens, is a possibility that requires research.

Methanogenesis predominates in the forestomachs of ruminants as the eructation reflex quickly removes any potentially danger to the integrity of the fermentative organ from gases released in fermentation of feed and maximises the potential for digestion of fibrous feeds by providing a sink for disposal of metabolic [H]. Microbial inoculation of feed in the rumen appears to occur to a major extent from established biofilms associated with feed particles and there is only minor modulation by immune tissue or secretions.

In the macropod forestomach, biofilms from the blind sac may be more like biofilms that occur in the lower gut of other non-ruminant mammals; during their development, these biofilms initially may obtain immune agents from the host that control their microbial species composition. The concept is that immune recognition benefits the macropod host by promoting mutualistic gut bacteria (Everett et al. 2004) as well as selecting against microbial species that are non-pathogenic, but could cause damage or disrupt homeostasis in the foregut (as proposed by Matzinger 2012).

To explain why macropods and horses rely on acetogenesis to dispose of metabolic [H], whereas ruminants rely on methanogenesis for the same purpose, it is argued that excessive gas production from the fermentation may illicit ‘damage signals’ and activate immune secretions in potentially affected organs to suppress the microbes responsible for releasing H₂ or CH₄. The effect of this immune modulation is to exclude methanogens from the caecum and haustral forestomach and create opportunities for acetogenic microbes to thrive, thus benefitting the animal by eliminating the damage to the gut tissues from excessive gas, while still maintaining mechanisms for efficient digestion of fibrous feeds.

The author declares no conflicts of interest.