Figure 8. Linear regression scatterplots indicating mean acid detergent lignin (ADL) degradation over 18 weeks for G. lucidum and P. ostreatus G1-G3 substrate grind consistencies

Discussion

The pretreatment of lignocellulosic substrates serves as a first step to the enhancement of potential feed-from-waste. Although all mushrooms used in this study delivered significantly improved results with reference to indigestible fraction degradation, the robusticity and reliability of the strains were key factors. G. lucidum has been shown to deliver similar results corresponding to numerous articles being published on its medicinal, lignolytic and biotechnological prowess [22,23,24]. Rapid colonisation of substrate bags was achieved with all strains completing colonisation within 7 days after inoculation. There were no significant differences observed in ADF, ADL or NDF reduction between the benchmark isolates P. ostreatus andG. lucidum (p > 0.05) although the benchmark strains did reduce observed parameters significantly more than then the control (p < 0.05). Therefore, only one grind consistency, G2, was selected for substrate when comparing the Ganoderma #1. It was interesting that no differences were observed as particle size was considered a key factor in studies performed by Batool and co-workers [25] when studying the effects of delignification of wheat straw using G. lucidum . It is believed that larger particle sizes did not allow for a large enough surface area for the colonisation of the fungus but by decreasing the particle size too much the substrate became anaerobic which diminishes the effect of the lignin degrading enzymes, which operate aerobically. A possible explanation could be that the aeration method on the substrate bags allowed for sufficient oxygen transfer negating any inhibitory effects due to insufficient oxygen transfer. Alternatively, the porosity of the particles the oxygen transfer will only become a significant factor at smaller particle consistencies. The reason for the choice to continue with G2 was simply the ease of grinding, reducing input costs, and the considerably smaller volume per weight ratio when considering transport, adding to the robusticity of the process. After the addition of the Ganoderma#1 in the repeat of the experiment (measuring only T0 and T18 and using G2 grind consistency) it was determined that Ganoderma #1 degraded the observed parameters significantly more than the control and the benchmark strains G. lucidum and P. ostreatus (p < 005).
There were no significant differences between the benchmark mushroomsG. lucidum and P. ostreatus tested in their ability to degrade NDF, ADF or ADL. Ganoderma #1 degraded the measured parameters significantly more than the benchmark strains, except for ADF, and all displayed increased degradation of parameters significantly when compared to the control. When viewed individually and observed from a total percentage ADF, NDF and ADL reduction in the total of each component rather than as a percentage of the total dry mass (Table 8, 9 and 10) the effects can be visualised more effectively. When considering that the lignin component of the lignocellulose matrix encapsulates 100% of the substrate, a 27% reduction in total lignin would indicate a significantly less recalcitrant substrate and increase accessibility to cellulose. When considering that not only degradation but also modification took place, where lignin strands are not necessarily broken down but also cut, the exposure of cellulose and hemicellulose to possible degrading microorganisms could be increased.
Although the analysis showed the effectiveness of the mushrooms in their degradation capabilities, it remains to be shown what influence they may have on the extremely complex ruminant digestive system given the antimicrobial properties of the Ganoderma genus. It will also be important to prove the digestibility through ruminant digestion rather than simply providing constituent numbers of the lignocellulose properties of the SSF product. The reduction of the lignin component by 4.76 % of the total DM of the substrate on average by all isolates tested equated to a reduction of nearly 25% of the total amount of lignin in the substrate. The cellulose fraction remained relatively intact and as a result the loss of gross energy available was only reduced by roughly 2% and crude protein increased by almost 30%. It has been shown that an enhancement in protein can be achieved by the treatment of fungi such as mushrooms [26]. Crude protein contents were significantly increased by fungal treatment and may have been a result of increased fungal biomass. Although the final amount of protein for the resulting SSF product was 5.6%, the increase of protein was not the primary goal of the experiment. Interestingly, although the P. ostreatus did produce the highest amount of protein the loss of energy through this process did remove a significant amount of available energy from the substrate.
Acacia mellifera contains a gross energy (GE) of 15.8 MJ/kg DM. This is a high energy potential when compared to Lucerne hay which contains 12.4 MJ/kg DM or sucrose with 15.6 MJ/kg [27]. The insignificant reduction in GE equates to higher available energy for ruminant digestion. P. ostreatus delivered a less significant delignification but yielded a significantly higher crude protein increase of 3.5% to 6.3%. The inherent problem with using P. ostreatus for the SSF purpose of this trial was the lack of overcoming the tree’s host defence as it can only survive saprophytically on decaying host plants instead of the parasitic abilities ofGanoderma spp.
The degradation of lignin occurs in a stepwise fashion and is unique to each mushroom’s degradation strategy. Lignin encapsulates cellulose and hemicellulose to provide protection from degradation and gives rigidity to the plant. This encapsulation is extremely resistant to microbial exploitation. The degradation strategy is mainly to biotransform lignin by cleaving the extended strands initially28. This would not be revealed analytically by determining the reduction of weight loss of the lignin component although the modification yields more accessibility to the fermentable components such as cellulose. This can be seen in large variation in the analysis data of the ADL fraction when compared to the NDF or ADF fractions’ confidence intervals. Where, stronger relationships would yield narrower confidence intervals as the variables in the model would account for a greater prediction of the population mean.
Solid-state fermentation is a valuable tool for the biotechnological sector and allows for numerous possibilities in the production of useful products from otherwise unusable wastes [15]. Acacia mellifera is abundant in central to northern South Africa grasslands/veld/etc., invasive and encroaching, it accounts for major losses in arable and grazing lands for farmers. Increased debushing efforts result in vast amounts of lignocellulose build-up and poses several problems for farmers, particularly as the primary means of disposal is the burning of the biomass [11]. South Africa has in recent years experienced severe drought and this has commanded the need for alternative sources of feed for domesticated livestock. Acacia mellifera has in the past been used as a form of feed addition due to its palatability and digestibility when still young and growing lusciously. The digestibility was still not acceptable but did serve as a short-term solution to starving animals. A practical solution would be to develop a pre-treatment method that is both economical and could be employed in robust and harsh environmental conditions. Fungi, such as mushrooms, are nature’s solution to decomposing and recycling nutrients back into the natural cycle, yet not all mushrooms are equally suited for biotechnological applications such as SSF. This research was aimed at using a local lignolytic basidiomycete capable of performing SSF and be able to withstand competition from cosmopolitan contaminant microbes and host plant defences. The need for a South African isolate was necessary to avoid possible contamination of the South African biodiversity. The Ganoderma #1 isolate proved to be remarkable in both its lignolytic abilities, and energy efficient, i.e., being able to digest significant amounts of lignin while leaving the desired cellulose fraction in-tact, and without utilising excessive amounts of available energy.
Considering the components of the resulting substrate product after SSF, it would be interesting to establish what fermentation and rumen digestion results could be obtained. Although Ganoderma #1 exceeded in degradation of indigestible components such as ADF, ADL and NDF this increased reduction could yield decreased fermentation ability due to excessive reductions of easily fermentable sugars not accounted for in this experiment. Pleurotus ostreatus showed promising degradation effects, although not as pronounced as the Ganodermaspecies, could yield a more increased permeability during rumen digestion due to increased protein production. Ganoderma lucidumdisplayed a middle of the field result not digesting as much asGanoderma #1 although slightly more than P. ostreatus . The NDF portion of the substrate was left more intact that that of theGanoderma species and with NDF being an important parameter for efficient digestion by ruminant microbes6 the P. ostreatus could yield improved fermentability.
The success of the SSF using mushrooms could only be verified by actual digestion by ruminant bacteria either in vivo or in vitro.The total gas production from fermentation would provide a wider view of the digestibility rather than looking at chemical components individually and forecasting theoretical digestibility. The ultimate goal of this process remains to transform an almost unfermentable substrate into a possible animal fodder with applications across a wide variety of livestock and industries.