Faecal samples analysed in this study were compared with soil collected from the same area to verify whether the surrounding surface soil differs from faecal matter regarding the microbial burden and enzymic activities. In addition, Nguni cow dung enabled a comparison of the wild herbivore faeces to that of a domestic herbivore fed on a fibrous diet.
Properties of faeces
The neutral to slightly alkaline pH of the giraffe and Nguni cow faeces (Table 1) was within the expected pH range of ruminants, which is typically between 6 and 8 (Artan et al. 1996; Moran 2005; Marãnón et al. 2006). Zebra and impala faeces had a slightly more alkaline pH (>8), possibly indicating the presence of alkali-tolerant microorganisms therein. Soluble chemical oxygen demand (sCOD) values indicated the potential availability of substrate in the faeces enabling growth of heterotrophic microorganisms. The soil samples had a more acidic pH and much lower moisture and soluble COD content than the faecal samples at the time of sampling, matching properties reported for infertile dry soils that have a low water binding capacity (Hartemink 2006, 2007). The highest sCOD of 339 mg/g was determined for Nguni cow faeces, while the sCOD values of the other faeces ranged between 57 and 80 mg/g. These values are within the expected sCOD range of 30–6000 mg/l reported for cattle in other studies (Marãnón et al. 2006; Abubakar and Ismail 2012). The higher moisture content in faecal samples indicated that the nature of the herbivore diet was mostly water binding fibrous material due to the uptake of grasses and leaves (Ziemer et al. 2012). As a result, for all the animals analysed in this study, faecal matter had higher moisture percentage values than matching soils samples. Based on the moisture content, pH and sCOD, faecal material clearly differed from the surrounding soil.
Analysis of enzymic activity
Microbial activity of samples was estimated through colorimetric assays that yield coloured reaction products once hydrolysis (FDA) or reduction (TTC) occurred. The fluorescein diacetate assay operates on the principle of hydrolytic release of two acetate groups via ester bond cleavage by free and membrane-bound hydrolytic enzymes, yielding the yellow-green coloured fluorescein that can be measured at 490 nm (Adam and Duncan 2001). FDA is a versatile substrate used for the detection of esterases within water bodies and individual cells (Battin 1997) and in soils (Green et al. 2006). However, other hydrolytic enzymes such as amylases can cleave its ester bonds as well (Lundgren 1981; Green et al. 2006). The FDA hydrolysis assay has also been utilised and recommended as an efficient method to estimate active cells within environmental samples (Swisher and Carroll 1980). The protocol employed in this study was optimised from Schnurer and Rosswall (1982) as suggested by Green et al. (2006) to establish the best possible assay conditions. Thus, acetone volumes used to terminate reactions were reduced to 4 % (v/v) and readings were taken within 30–60 min of termination to enable stable results (Green et al. 2006). As the FDA assay can be problematic due to abiotic cleavage caused by media components (Clarke et al. 2001; Wanandy et al. 2005), this was accounted for through use of an appropriate buffer and controls accounting for abiotic cleavage.
The FDA assay demonstrated that faecal samples from wild herbivores had higher hydrolase activity than soil controls and cow faeces, which is expected on microbiological grounds (Fig. 1a). In addition, samples incubated under shaker conditions showed higher activity than those incubated statically (Fig. 1a). Although this is contrary to the findings of Green et al. (2006) who reported that shaking decreased the amount of fluorescein released in soil samples, it confirms recommendations by Swisher and Carroll (1980) and Schnurer and Rosswall (1982) that shaking during incubation increases hydrolytic activity due to improved homogenisation. In addition, shaking improved the activity of hydrolytic microorganisms (Bozic et al. 2011) and substrate distribution (Juergensmeyer et al. 2007). The fact that a control with autoclaved faeces yielded no measurable activity in the FDA assay confirmed that its hydrolysis depended on the presence of hydrolytically active microorganisms or their enzymes in the faeces. Furthermore, the higher hydrolytic activity in faecal samples suggested that more hydrolase producing microorganisms were present in faeces than in soil as was confirmed by the plate counts (Table 2). Again, this is not unexpected as an increased degree of FDA hydrolysis relies on the presence of metabolically active microorganisms (Chrzanowski et al. 1984).
The TTC assay operates on the principle of reduction of colourless 2,3,5-triphenyltetrazolium chloride by active dehydrogenases to produce the corresponding coloured triphenylformazan (TPF). The water insoluble TPF can be extracted from samples using solvents such as methanol, butanol and ethanol and is optimally measured at 485 nm (Stevenson 1959). The assay can be used to determine cell viability (Tergendy et al. 1967) and for the analysis of microbial activity present in soils and other samples (Stevenson 1959). Like the FDA assay, the TTC assay estimates cell activity through enzyme activity. Due to its somewhat lower sensitivity, the TTC assay requires sample incubation periods of at least 24 h (Ishikawa et al. 1995). In addition, in the presence of O2 as a competing electron acceptor the dehydrogenase activity is potentially underestimated (Von Mersi and Schinner 1991). Therefore, samples analysed by the TTC assay in this study were incubated statically for 1 week. The TTC results are in good agreement with the findings of the FDA assay (Fig. 1). Addition of glucose confirmed the presence of glucose utilising microorganisms by leading to higher overall dehydrogenase activity due to increased microbial biomass (Fig. 1b). This is expected since gut microorganisms in ungulates ultimately break down cellulose to glucose. The high dehydrogenase and hydrolase activity of zebra faeces in the presence of a slightly elevated pH might indicate the presence of microorganisms therein able to function at higher than neutral pH, possibly indicating the presence of enzymes with potential for use in industrial applications requiring alkaline conditions (Horikoshi 1999). Comparison of sample pH values (Table 1) and TPF formation rates (Fig. 1b) indicates that increased TPF formation took place at neutral to slightly alkaline pH (i.e. pH > 7), confirming previous studies showing that the activity of electron transport systems (ETS)—including dehydrogenase activity—is enhanced within a pH range of 7.4–8 (Trevors 1984).
Microbial counts
Viable counts in faecal samples were mostly in the range of 108 cells per gram dry weight, with each faecal sample containing all targeted hydrolase producers although strictly anaerobic hydrolytically active bacteria were not quantified (Table 2). The counts for soil control samples were generally at least 1–2 logs lower than the counts in the corresponding faecal samples. In addition, clear differences in the proportions of specific hydrolase producers were evident (Fig. 2). Gong (2007) reported total and viable microscopic counts for cattle dung of about 1011 cfu/g (dry weight) and a matching plate count for mesophilic aerobic and facultatively anaerobic bacteria of about 1010 cfu/g, with proteolytic bacteria present at about 108 cfu/g and both lipolytic, amylolytic and cellulolytic bacteria present at about 109 cfu/g. Viable plate counts established in this study for Nguni cow faeces were lower than those reported by Gong (2007), although similar relative proportions were observed for the hydrolase producers with the exception of cellulolytic microorganisms. The difference in viable counts per g of faeces might be due to the shorter incubation time for agar plates used and the typically poorer diet of the Nguni cow (Tada et al. 2013).
With the exception of lipolytic esterase producing organisms, faecal samples from impala and giraffe showed similar proportions of amylase, cellulose and protease-producers (Fig. 2). Both impala and giraffe, ruminants as opposed to the non-ruminant zebra, had relative proportions of 91 and 95 % for amylase producers, 89 and 93 % of protease producers and fairly similar percentages of cellulase producers (52 and 74 %). Zebra in turn had the highest proportion of esterase producers (72 %) and the lowest proportion of protease and cellulase producers (17 and 21 %) amongst the three ungulates. In comparison, Nguni faeces was similar to zebra faeces with a fairly high proportion of amylase producers (46 %), a low proportion of protease producers (4.3 %) and an almost identical proportion of cellulose producers (22 %) present. Nguni faeces displayed lower hydrolytic and dehydrogenase activity compared to other ungulates, which could be due to lower proportions of hydrolase producers present and the different digestive system. These findings are consistent with previous reports in the literature suggesting that cellulolytic microorganisms when in competition with other specialists are present at low proportions (Witten and Richardson 2003). The different digestive systems present in ruminants (for example giraffe) and hindgut digesters such as zebra might result in different microbial communities.
A study of the giraffe rumen microbiome by Roggenbuck et al. (2014) showed via sequence analysis that in addition to many unknown bacteria, strictly anaerobic bacteria constitute a large proportion of the rumen community and that diet might influence the composition of this community.
The apparent differences in microbial colony counts and diversity of hydrolytically active bacteria within faeces from different herbivores (Table 2) might be due to their different feeding habits. Zebra and Nguni cattle are grazers (Odadi et al. 2011) whilst giraffe is a browser and impala is a mixed feeder (grazer and browser) (Codron et al. 2005). In Nguni cow and zebra faeces the proportion of esterase producers was higher than in faeces of giraffe and impala (Fig. 2) while the opposite applies to the proportion of cellulose producers which was lower in cow and zebra faeces than in faeces from giraffe and impala. Grazers feed more on grasses that are considered less nutritious than other forage and grazing is a lengthier process than browsing (Udén and Van Soest 1982). Zebra faeces, however, displayed a higher hydrolytic activity than Nguni cow faeces (Fig. 1a) which may be due to the difference in their digestive systems and the microbial community present. As large sized ruminants, cows have a longer digestion than non-ruminants and require more grazing time. Hydrolytic microorganisms from cattle form biofilms on forage substrates within the rumen to efficiently hydrolyse polymeric substrates (McSweeney et al. 1999) with an efficient digestion of biopolymers such as cellulose taking place in the foregut, mostly facilitated by anaerobic microorganisms. Movement of cud to the hindgut is usually required for secondary digestion. As a result, lower counts for aerobic microorganisms and hydrolase producers are expected in cow faeces (Mackie 2002). Although grass diets of cow and zebra are similar, zebra as hindgut fermenters possess an advantage by feeding on greater forage variety for shorter periods (Odadi et al. 2011). Zebra living in the local nature reserve probably had access to a more diverse diet than the Nguni cow kept at a local farm.
This study indicates that relative proportions of hydrolase producers in faeces—even though strictly anaerobic hydrolase producers were not quantified—are mostly similar for comparable feeding habits while hydrolytic activities and microbial loads can differ. This might be a result of different digestive systems and digestion times. The data for cattle and zebra indicate somewhat higher numbers of viable microorganisms in equine than in bovine faeces possibly due to shorter retention time (Odadi et al. 2011) resulting in less efficient polymer hydrolysis. As digestion occurs within the hindgut, forage is usually defecated without being properly hydrolysed resulting in more faecal shedding in zebra (Mackie 2002) than in cattle, as indicated by the results in this study.
Impala and giraffe appear to be similar regarding the relative proportions of protease, cellulose and amylase producers. As ruminants, they have a more complex digestive system than zebra and as browsers a more diverse diet. The impala in this study might have fed on a diet similar to the diet of giraffe, comprising of more browse forage than grasses, resulting in mostly similar relative hydrolase producer proportions. The smaller body size of impala means that digestion time is shorter compared to cows and faecal microbial numbers may therefore be higher (Gordon 2003).
The presence of proportionally more lipolytic esterase producers in zebra and Nguni cow than in impala and giraffe faeces might be due to the varying lipid content of plant species (Hadley and Rosen 1974) included in their diet. The presence of protease producers in faeces—although at a lower relative proportion in zebra and cow faeces—was expected, as proteases are present in the majority of heterotrophic microorganisms. Similarly, cellulose and amylase producing microorganisms were observed which is expected based on the presence of large amounts of cellulose and starch in leaves and grasses (Ben-Shahar and Coe 1992). Apart from the hydrolases targeted in this study, other hydrolytic enzymes which are of interest for industry (Kirk et al. 2002) such as xylanases and hemicellulases are typically detected in faecal samples of ungulates (Fon and Nsahlai 2012).
Microbial interactions in the intestinal systems of these herbivores are similar to anaerobic bioreactors utilizing cellulosic material as substrate, with similar hydrolytically active bacteria documented as members of the anaerobic food chain in artificial (bioreactors) and natural systems (digestive tract of herbivores) (Krakat et al. 2011; Morrison et al. 2009).