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.