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\begin{document}

\title{The application of solid-state fermentation using mushrooms for the
production of animal feed}



\author[1]{E.D Cason}%
\author[2]{ C. Rothmann}%
\author[2]{L.A Rothmann}%
\author[2]{B.C Viljoen}%
\affil[1]{University of the Free State}%
\affil[2]{University of the Free State - Bloemfontein Campus}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The increasing growth of agro-industrial activity resulting in excessive
amounts of agri-waste has led to the accumulation of a large quantity of
lignocellulosic residues all over the world, in particular,
deforestation initiatives for the removal of invasive trees in South
Africa. These lignocellulosic residues are rich in energy resources
consisting of a mixture of natural polymers based on lignin, cellulose
and hemicellulose. The use of lignolytic fungi such as mushrooms in
solid-state fermentation could sufficiently degrade the indigestible
lignocellulosic components and add medicinal and nutritional value to
otherwise unusable, high-energy waste material. The digestive type of
animal for which the potential feed is developed must be identified and
considered before deciding on the bioconversion method and process,
since the outcomes for obtaining potentially high-quality feeds for
non-ruminant and ruminant animals are different. The current study
presents the data of the bioconversion of lignocellulosic substrate
using solid-state fermentation with the edible and medicinal mushrooms,
Ganoderma lucidum, Pleurotus ostreatus, and a possible new species, to
increase digestibility and nutritional value to be applied as ruminant
animal feed. The solid-state fermentation process was optimised and the
resulting product analysed for the degradation of the lignocellulosic
components. Results indicated that the solid-state fermentation duration
and mushroom species were key components in achieving significant
degradation. Data obtained after 18 weeks of degradation indicated a
significant (p \textless{} 0.05) reduction in the acid detergent fibre,
acid detergent lignin and neutral detergent fibre fractions of the
biomass, with up to 20\% reduction in indigestible components.%
\end{abstract}%



\sloppy


\textbf{Manuscript Title:}

The application of solid-state fermentation using mushrooms for the
production of animal feed

\textbf{Authors:}

Christopher Rothmann\textsuperscript{a,c}, Dr. Lisa
Rothmann\textsuperscript{b}, Bennie Viljoen\textsuperscript{c}, Errol
Duncan Cason\textsuperscript{a}

\textsuperscript{a} Department of Animal Sciences, University of the
Free State, Bloemfontein South Africa

\textsuperscript{b} Department of Plant Sciences, University of the Free
State, Bloemfontein, 9300, South Africa

\textsuperscript{c} Department of Microbiology and Biochemistry,
University of the Free State, Bloemfontein South Africa

\textbf{Keywords:}

Biotechnology, Fermentation, Solid-State Fermentation,
Rumen,\emph{Ganoderma} , \emph{Pleurotus,} Ruminant Digestion,
\emph{Acacia mellifera,} Animal Feed, Pretreatment

\textbf{Corresponding Author}

Prof Errol Cason, Office LG3.G02A, Agricultural Building, Department of
Animal Sciences, University of the Free State, 205 Nelson Mandela Drive,
Park West, Bloemfontein, 9301, South Africa. Email Address:
CasonED@UFS.ac.za

\textbf{Abbreviations}

ADF - Acid Detergent Fibre

ADL - Acid Detergent Lignin

ANOVA - Analysis of variance

CP - Crude Protein

DM - Dry Matter

GE - Gross energy

ITS - Internal Transcribed Spacer

LSD - Least Significant Difference

NDF - Neutral Detergent Fibre

PDA - Potato Dextrose Agar

SD - Standard deviation

SEM - Standard error of the mean

SSF - Solid-State Fermentation

\textbf{The application of solid-state fermentation using mushrooms for
the production of animal feed}

C. Rothmann\textsuperscript{a,c}, L.A Rothmann\textsuperscript{b}, B.C
Viljoen\textsuperscript{c}, E.D Cason\textsuperscript{a}

\textsuperscript{a} Department of Animal Sciences, University of the
Free State, Bloemfontein South Africa

\textsuperscript{b} Department of Plant Sciences, University of the Free
State, Bloemfontein, 9300, South Africa

\textsuperscript{c} Department of Microbiology and Biochemistry,
University of the Free State, Bloemfontein South Africa

\textbf{Abstract}

The increasing growth of agro-industrial activity resulting in excessive
amounts of agri-waste has led to the accumulation of a large quantity of
lignocellulosic residues all over the world, in particular,
deforestation initiatives for the removal of invasive trees in South
Africa. These lignocellulosic residues are rich in energy resources
consisting of a mixture of natural polymers based on lignin, cellulose
and hemicellulose. The use of lignolytic fungi such as mushrooms in
solid-state fermentation could sufficiently degrade the indigestible
lignocellulosic components and add medicinal and nutritional value to
otherwise unusable, high-energy waste material. The digestive type of
animal for which the potential feed is developed must be identified and
considered before deciding on the bioconversion method and process,
since the outcomes for obtaining potentially high-quality feeds for
non-ruminant and ruminant animals are different. The current study
presents the data of the bioconversion of lignocellulosic substrate
using solid-state fermentation with the edible and medicinal
mushrooms,\emph{Ganoderma lucidum, Pleurotus} \emph{ostreatus} , and a
possible new species, to increase digestibility and nutritional value to
be applied as ruminant animal feed. The solid-state fermentation process
was optimised and the resulting product analysed for the degradation of
the lignocellulosic components. Results indicated that the solid-state
fermentation duration and mushroom species were key components in
achieving significant degradation. Data obtained after 18 weeks of
degradation indicated a significant (p \textless{} 0.05) reduction in
the acid detergent fibre, acid detergent lignin and neutral detergent
fibre fractions of the biomass, with up to 20\% reduction in
indigestible components. This increase in digestibility could contribute
to to increased energy availability for ruminant animals.

\textbf{Keywords:}

Biotechnology, Fermentation, Solid-State Fermentation,
Rumen,\emph{Ganoderma} , \emph{Pleurotus,} Ruminant Digestion,
\emph{Acacia mellifera,} Animal Feed, Pretreatment

\subsubsection*{Introduction}

{\label{introduction}}

Growth and expansion in the agricultural and food sectors results in the
generation and accumulation of a vast amount of agro-industrial waste
every year. Agro-industrial waste is defined as ``the waste generated
during the industrial processing of agricultural or animal products or
the waste obtained from agricultural activities'' by Mirabella and
co-workers {[}1{]}. Environmentally friendly or acceptable disposal
methods of agricultural wastes and residues pose a significant
scientific problem, especially when considering the chemical properties,
recalcitrance, and abundance of lignocellulose. An increase of interest
was also noted for alternative treatments other than expensive physical
or chemical treatments, such as the use of white-rot fungi through
solid-state fermentation to degrade lignin components in biomass,
supported by an amassing number of published research on this subject
{[}2{]}. The use of macrofungi, such as mushrooms, which naturally occur
in nature as wood or organic litter decomposers, excrete nonspecific
oxidative enzymes that degrade lignocellulosic materials, and thereby
offer solutions that are being widely and readily applied for the
biotransformation of these materials. Equally important, these organisms
have shown efficient bioconversion and degradation of various types of
agro-industrial/forestry by-products with low or no economic value to
edible biomass {[}3{]}.

Any biotechnological method in which living organisms are cultivated on
non-soluble or solid material in the absence (or near absence of free
water) is recognised as solid-state fermentation (SSF) {[}4{]}.
Solid-state fermentation has been shown to be the most effective method
to harness the power of these macrofungi for the purposes of
delignification {[}4{]}, conversion {[}3{]} and nutritional value
addition {[}5{]}. White-rot fungi have been shown to digest or degrade
lignin and sequentially break down the lignocellulose complexes,
increasing nutrient availability for microbial fermentative utilisation
during rumen digestion {[}6{]}. Basidiomycetous white-rot fungi, such
as\emph{Pleurotus ostreatus,} digest lignocellulose in anticipation of
producing fruiting structures (mushrooms) and display a unique strategy
to colonise and modify the substrate in such a way that readily
metabolizable cellulose is available when fruit bodies are produced.
During mycelial growth they sequentially produce enzymes that degrade or
modify lignin and change the enzyme target towards degradation of
cellulose and hemicellulose during fruiting. Stopping this process
before fruiting results in an organic substrate with fewer
hemicellulose-lignin bonds and subsequently increases accessibility of
cell-wall carbohydrates for rumen microbes to digest. The ultimate
effectiveness of the fungal pretreatment hinges on factors such as
biochemical characteristics of the material, the choice of fungal strain
used and the amount of time of the fungal treatment {[}7{]}.

The encroaching bush \emph{Acacia mellifera,} locally known as Swarthaak
(Figure 1), has formed vast impenetrable thickets in the central to
northern parts of southern Africa, detrimentally impacting grazing
forage {[}8,9,10,11{]}. Debushing efforts have somewhat alleviated the
physical presence, yet mechanical removal of the encroachment only
serves as temporary solutions. In order to increase sustainability,
Lukomska and co-workers {[}11{]} refer to the necessity to find an
alternative use for harvested biomass to offset the short-term income
risk debushing holds for farmers. Converting harvested encroaching
biomass to livestock, especially ruminant, fodder could provide
sustainable solutions to multiple problems. The primary goal of the SSF
design of this research is for maximal reduction or bioconversion of
lignin with minimal loss in cellulose and hemicellulose as shown in
Figure 2.~This study presents a SSF method for the selective
delignification of \emph{Acacia mellifera} using basidiomycetous fungi,
i.e., mushrooms, as the biological agents.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} A large \emph{Acacia mellifera} tree with a
surrounding impenetrable thicket of seedlings (Picture by author).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} The constituents of the carbohydrate fraction of
woody plant residues and the inherent fermentability of the basic
building blocks.

\subsection*{}

{\label{section}}

\subsection*{Materials and Methods}

{\label{materials-and-methods}}

\subsubsection*{Fungal strain selection}

{\label{fungal-strain-selection}}

\emph{Pleurotus ostreatus} (AM-G269) and \emph{Ganoderma
lucidum}(AM-M177) were purchased from Aloha Medicinals (Carson City,
Nevada, USA). These strains were selected due to proven cultivation
ability and for international comparability. A further strain search was
launched to locate and isolate South African strains for assessment. A
potential newly discovered \emph{Ganoderma} \#1 isolate was included in
this study due to its inherent higher optimal growth temperature and
only tested at time 0 and after 18 weeks on the G2 substrate
consistency. To avoid possible spreading of spores into the environment
the study was designed to avoid fruiting cycles of \emph{Ganoderma} as
the mycelial state of\emph{Ganoderma} does not possess the ability to
effectively infect hosts non-intentionally.

\subsubsection*{Substrate for SSF}

{\label{substrate-for-ssf}}

\emph{Acacia mellifera} was chosen as the preferred substrate due to
excessive volumes available in central South Africa. Substrate was
milled to three different particle sizes to elucidate possible
variations due to the available surface area of the substrate and
distinguished by G3 (rough chips, 2-5 cm), G2 (medium chips, 1-2 cm) and
G1 (fine chips, <\selectlanguage{english}1 cm). The woody substrate was allowed to `rest' for
two weeks before use to circumvent any passive host-immunity to the
mushroom strains. \emph{Ganoderma} \#1 was included in a replicate
experiment after the completion of the benchmark strains and only G2 was
selected as the variable grind consistency.

\subsubsection*{Substrate cultivation
bags}

{\label{substrate-cultivation-bags}}

Substrate was soaked in demineralised water for 24 hours for hydration.
The hydrated substrate was subsequently drained until no free running
water was observed. Substrate was packed into polypropylene bags with
0.45 \selectlanguage{greek}µ\selectlanguage{english}m pore size filter patches to contain a total of 400 g
hydrated\emph{A. mellifera} wood chips in the three different size
categories, i.e.,. All substrate bags were autoclaved for 90 minutes at
121 \selectlanguage{ngerman}°C. Cultivation bags were performed in duplicate for a total of 20
weeks with sample bags being chosen at random every two weeks for
analysis.

\subsubsection*{Mushroom cultures}

{\label{mushroom-cultures}}

The purchased mushroom cultures were received on Difco\selectlanguage{english} Potato Dextrose
Agar (PDA) slants. Upon reception the cultures were transferred
aseptically to fresh PDA plates and allowed to grow for seven days
before being used as spawn inoculum. All species applied were previously
characterised on genomic level using the Internal Transcribed Spacer
(ITS gene region.

\subsubsection*{Mushrooms spawn
production}

{\label{mushrooms-spawn-production}}

Sorghum grains were soaked for 24 hours for hydration. The grains were
removed from the soaking water and surface-dried by rolling on a towel
until no longer visibly wet on the surface. The grains were then placed
in polypropylene cultivation bags with air with 0.45 \selectlanguage{greek}µ\selectlanguage{english}m pore size
filters and sterilised by autoclaving for 90 mins at 121 \selectlanguage{ngerman}°C. After being
cooled to room temperature the bags were inoculated by adding a 1 x 1 cm
square of PDA agar covered with mushroom mycelium. Bags were then
incubated at 25 °C until fully colonised.

\subsubsection*{Substrate inoculation}

{\label{substrate-inoculation}}

Each 400 g substrate bag was inoculated with 40 g of sorghum grain
mushroom spawn and shaken to distribute inoculum. The substrate bags
were placed in a growth room at 25 °C and humidity of 85\% (Figure 3).
Control samples were inoculated with the same method but immediately
placed in an oven at 80 °C to dry completely and milled to a fine
powder.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Substrate bags colonized by \emph{G. lucidum} 7 days
after inoculation

\subsubsection*{Fibre content analysis}

{\label{fibre-content-analysis}}

Samples were analysed and calculations performed for neutral detergent
fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL)
using the ANKOM 220 Fibre Analyzer (ANKOM Technology Corporation, NY,
USA) according to the Van Soest and co-workers (1991) method {[}12{]}.
NDFom-NDF was not assayed with a heat stable amylase and was expressed
exclusive of residual ash. ADFom-ADF expressed exclusive of residual
ash. Lignin (sa)-Lignin was determined by solubilization of cellulose
with sulphuric acid.

The ADF content of samples was calculated as follows:

ADF (g/kg DM) = Sample weight after boiling (g DM) -- Ash weight (g DM)/
ADF (g/kg DM) / Weight of sample (g DM) \selectlanguage{ngerman}× 1 000

The NDF content of samples was calculated as follows:

NDF (g/kg DM) = Sample weight after boiling (g DM) -- Ash weight (g DM)
/ Weight of sample (g DM) × 1 000

The ADL content of samples was calculated as follows:

ADL (g/kg DM) = Sample weight (g DM) -- Ash weight (g DM) / Weight of
sample (g DM) × 1~000

\subsubsection*{Crude Protein}

{\label{crude-protein}}

Crude protein (CP) was determined for the raw substrate and the SSF
product in triplicate at each selected stage (time 0 and time 18 weeks).
The nitrogen (N) content of NDF residues was analysed by combustion
assay {[}13{]}. One-gram (1 g) samples were weighed accurately to
determine the CP content by inserting it into a Leco Nitrogen analyser
and the total N content determined on combustion in oxygen {[}14{]}. A
factor of 6.25 was used to convert the N content of the samples to CP
content {[}13{]}.

\subsubsection*{Gross Energy
determination}

{\label{gross-energy-determination}}

One gram (1 g) of the samples were analysed in triplicate to determine
the GE content according to the procedures described by the AOAC
{[}15{]}.

\subsubsection*{Factorial design and data
analysis}

{\label{factorial-design-and-data-analysis}}

Data processing and analyses were performed with R version 4.0.2
{[}16{]} within R Studio version 1.2.5042 {[}17{]}. Data were imported
using the `gsheet` package {[}18{]}. Data exploration, wrangling and
visualisation were conducted using the `tidyverse` package {[}19{]}.
Analysis of variance (ANOVA; \selectlanguage{greek}α \selectlanguage{english}= 0.05) of the data were performed using
base R functions. ANOVA was applied to determine if mushroom species
(\emph{Ganoderma lucidum, Ganoderma} \#1 \emph{and Pleurotus ostreatus}
) and grind (sieve sizes of 2 to 5 cm, 1 to 2 cm and \textless{} 1 cm,
for grind 3, 2 and 1, respectively) affected dependent variables (ADF,
ADL and NDF) from the first and last sampling period, 0 to 18 weeks.
Means were separated using the Least Significant Difference (LSD) test
function from the `agricolae` package {[}19{]}. A linear regression
analysis was applied to identify if any associations existed between the
volume of the dependent variables over the 18-week sampling period.
Pearson correlation coefficient was applied in the scatter plots and
plotted with the `ggpubr` package {[}21{]} to determine the strength of
the associations presented.

\subsection*{Results}

{\label{results}}

\subsubsection*{Crude Protein}

{\label{crude-protein-1}}

Crude protein (CP) determination of the various substrates was performed
after 18 weeks of SSF (Figure 4). The undigested control yielded mean CP
concentrations (g/kg DM) of 39.98 (\selectlanguage{ngerman}±3.31), \emph{Ganoderma lucidum}55.58
(±1.13), \emph{Pleurotus ostreatus} 61.48 (±1.46) and\emph{Ganoderma}
\#1 isolate yielded 53.25 (±0.38) as shown in Table 1.

\textbf{Table 1:} Crude protein (CP) concentrations (g/kg DM) of
mushroom treatments after 18 weeks solid-state fermentation of S:
undigested \emph{Acacia mellifera} control, M1: \emph{Pleurotus
ostreatus} substrate, M2: \emph{Ganoderma lucidum} substrate,
M3:\emph{Ganoderma} \#1 substrate.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Treatment} & \textbf{Average} & \textbf{SD} &
\textbf{SEM}\tabularnewline
\midrule
\endhead
S & 39,98 & 3,3 & 12,07\tabularnewline
M1 & 55,68 & 1,13 & 49,46\tabularnewline
M2 & 61,48 & 1,46 & 42,2\tabularnewline
M3 & 53,25 & 0,38 & 140,18\tabularnewline
\bottomrule
\end{longtable}

SSF: solid-state fermentation, SD: Standard deviation, SEM: Standard
error of the mean\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Crude protein content (g/kg DM) of substrate after 18
weeks of solid-state fermentation using \emph{G. lucidum} , \emph{P}
.\emph{ostreatus} and \emph{Ganoderma} \#1 compared to an undigested
control.

\subsubsection*{Gross Energy~}

{\label{gross-energy}}

Gross energy (GE) determination of the various substrates was performed
after 18 weeks of SSF (Figure 5). The undigested control yielded a GE
content of 15.79 MJ/kg DM (\selectlanguage{ngerman}±0.55), \emph{Ganoderma lucidum} 15.18 MJ/kg
DM (±0.18), \emph{Pleurotus ostreatus} 13.28 MJ/kg DM (±0.18)
and\emph{Ganoderma} \#1 isolate yielded 15.54 MJ/kg DM (±0.46) as shown
in Table 2.

\textbf{Table 2:} Gross energy (GE) concentrations (MJ/kg DM) of
mushroom treatments S: Undigested \emph{Acacia mellifera,}
M1:\emph{Pleurotus ostreatus} substrate, M2: \emph{Ganoderma
lucidum}substrate, M3: \emph{Ganoderma} \#1 substrate after 18 weeks
solid-state fermentation.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Treatment} & \textbf{Average} & \textbf{SD} &
\textbf{SEM}\tabularnewline
\midrule
\endhead
S & 15,79 & 0,55 & 0,32\tabularnewline
M1 & 15,81 & 0,18 & 0,1\tabularnewline
M2 & 13,28 & 0,18 & 0,11\tabularnewline
M3 & 15,54 & 0,46 & 0,26\tabularnewline
\bottomrule
\end{longtable}

SSF: solid-state fermentation, SD: Standard deviation, SEM: Standard
error of the mean.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Gross Energy content (MJ/kg DM) of substrate after 18
weeks of solid-state fermentation using \emph{G. lucidum} , \emph{P}
.\emph{ostreatus} and \emph{Ganoderma} \#1 compared to an undigested
control.

\subsubsection*{Quantifying solid-state
fermentation}

{\label{quantifying-solid-state-fermentation}}

Analysis of variance (ANOVA) indicated time of experimental sampling as
the only main effect resulting in significant differences (p \textless{}
0.05) for the mean ADF (Table 3) and mean NDF (Table 4). However,
mushroom species and time of experimental sampling contributed to
significant differences in the mean ADL (Table 5). Means separation
indicated all time 0 samples held the highest concentration for all
measured degradation parameters, ADF, NDF and ADL. After 18 weeks of
solid-state fermentation, all concentrations had collectively reduced by
11.66, 14.84 and 4.76 \% (equating to g per 100 g dry matter; DM) for
mean ADF, NDF and ADL, respectively (Table 6). The mushroom species
which had supreme ability to degrade ADL was \emph{Ganoderma \#} 1,
where the lowest mean ADL (16.47 g per 100 g DM) was recorded.
Consequently,\emph{Pleurotus ostreatus} and \emph{Ganoderma lucidum} had
reduced abilities to degrade ADL compared to that of
\emph{Ganoderma}\#1\emph{,} although all mushroom species held the
ability to significantly degrade ADL through SSF collectively (Table 6).
When viewed individually (Table 7), as indicated by means
separation,\emph{Ganoderma} \#1 showed increased degradation of all
measured parameters, with the exception of ADF. Additionally,
\emph{Ganoderma}\#1 performed significantly greater in degradation
capacity when compared to benchmark strains \emph{G. lucidum} and
\emph{P. ostreatus}(p \textless{} 0.05) which did not degrade parameters
significantly when compared between each other, although did degrade
parameters significantly more than the control (p \textless{} 0.05).

\textbf{Table 3.} Analysis of variance for mean acid detergent fibre
(ADF) across three mushroom species (\emph{Ganoderma lucidum,
Ganoderma}\#1 and \emph{Pleurotus ostreatus} ), with three substrate
grind consistency G2, where samples were taken from experiment
initiation (week 0) to competition (week 18).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{SOV} & \textbf{DF} & \textbf{SS} & \textbf{MS} & \textbf{F
value} & \textbf{Pr (\textgreater{}F)}\tabularnewline
\midrule
\endhead
Mushroom & 2 & 1,80 & 0,90 & 0,39 & 0,690\tabularnewline
Time & 1 & 407,50 & 407,50 & 177,49 & 9,6E-07\tabularnewline
Residuals & 8 & 18,40 & 1,64 & &\tabularnewline
\bottomrule
\end{longtable}

SOV: Source of variation, DF: Degrees of freedom, SS: Sum of squares,
MS: Mean square.

\textbf{Table 4.} Analysis of variance for mean neutral detergent fibre
(NDF) across two mushroom species (\emph{Ganoderma lucidum,
Ganoderma}\#1 and \emph{Pleurotus ostreatus} ), with three substrate
grind consistency G2, where samples were taken from experiment
initiation (week 0) to competition (week 18).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{SOV} & \textbf{DF} & \textbf{SS} & \textbf{MS} & \textbf{F
value} & \textbf{Pr (\textgreater{}F)}\tabularnewline
\midrule
\endhead
Mushroom & 2 & 18,91 & 9,45 & 0,7839 & 0,49\tabularnewline
Time & 1 & 660,23 & 660,23 & 54,7518 & 7,62E-05\tabularnewline
Residuals & 8 & 96,47 & 12,06 & &\tabularnewline
\bottomrule
\end{longtable}

SOV: Source of variation, DF: Degrees of freedom, SS: Sum of squares,
MS: Mean square.

\textbf{Table 5.} Analysis of variance for mean acid detergent lignin
(ADL) across two mushroom species (\emph{Ganoderma lucidum,
Ganoderma}\#1 and \emph{Pleurotus ostreatus} ), with three substrate
grind consistency G2, where samples were taken from experiment
initiation (week 0) to competition (week 18).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{SOV} & \textbf{DF} & \textbf{SS} & \textbf{MS} & \textbf{F
value} & \textbf{Pr (\textgreater{}F)}\tabularnewline
\midrule
\endhead
Mushroom & 2 & 1,02 & 0,51 & 0,41 & 0,679\tabularnewline
Time & 1 & 67,97 & 67,97 & 53,96 & 8,03E-05\tabularnewline
Residuals & 8 & 10,08 & 1,26 & &\tabularnewline
\bottomrule
\end{longtable}

SOV: Source of variation, DF: Degrees of freedom, SS: Sum of squares,
MS: Mean square.

\textbf{Table 6.} Means separation through Fisher's Least Significant
Difference for mean acid detergent fibre (ADF) (\% dry mass), neutral
detergent fibre (NDF) and acid detergent lignin (ADL) between two
experimental time units, from start (week 0) to completion (week 18) of
all benchmark treatments combined.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Time (Weeks)} & \textbf{Mean ADL} & \textbf{Mean NDF} &
\textbf{Mean ADF}\tabularnewline
\midrule
\endhead
0 & 21,69\textsuperscript{a} & 79,40\textsuperscript{a} &
64,24\textsuperscript{a}\tabularnewline
18 & 17,20\textsuperscript{b} & 64,56\textsuperscript{b} &
52,58\textsuperscript{b}\tabularnewline
LSD & 1,49 & 4,62 & 2,02\tabularnewline
\bottomrule
\end{longtable}

LSD: Least significant difference, ADF: Acid detergent fibre, NDF:
Neutral detergent fibre, ADL: Acid detergent lignin. Mean values
followed by the same letter do not differ significantly according to
Fisher's Least Significant Difference (LSD, \selectlanguage{greek}α \selectlanguage{english}= 0.05).

\textbf{Table 7.} Means separation through Fisher's Least Significant
Difference for mean acid detergent fibre (ADF), neutral detergent fibre
(NDF) and acid detergent lignin (ADL) of substrates M1 (\emph{P.
ostreatus} ), M2 (\emph{G. lucidum} ), M3 (\emph{Ganoderma} \#1) and S
(untreated \emph{Acacia mellifera} ) between all fungal treatments
expressed as percentage (\%) of dry mass (DM).\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Treatment} & \textbf{Mean ADL} & \textbf{Mean NDF} &
\textbf{Mean ADF}\tabularnewline
\midrule
\endhead
M1 & 17,71\textsuperscript{a} & 68,63\textsuperscript{a} &
53,53\textsuperscript{a}\tabularnewline
M2 & 17,44\textsuperscript{a} & 62,38\textsuperscript{a} &
52,33\textsuperscript{a}\tabularnewline
M3 & 16,47\textsuperscript{b} & 62,69\textsuperscript{b} &
51,89\textsuperscript{a}\tabularnewline
S & 22,02\textsuperscript{c} & 79,51\textsuperscript{c} &
64,56\textsuperscript{b}\tabularnewline
LSD & 0,71 & 3,16 & 1,32\tabularnewline
\bottomrule
\end{longtable}

LSD: Least significant difference, ADF: Acid detergent fibre, NDF:
Neutral detergent fibre, ADL: Acid detergent lignin. Mean values
followed by the same letter do not differ significantly according to
Fisher's Least Significant Difference (LSD, \selectlanguage{greek}α \selectlanguage{english}= 0.05).

\subsubsection*{Total fibre reduction}

{\label{total-fibre-reduction}}

In order to determine degradation of the lignocellulose complex for the
duration of the experiment, linear regression scatterplots and Pearson
correlation coefficients (R) were conducted. All Pearson correlation
coefficients, with the exception of mean ADL percentage at grind 1
of\emph{G. lucidum} (p \textasciitilde{} 0.053), were significant (\selectlanguage{greek}α \selectlanguage{english}=
0.05). Solid-state fermentation across grind consistency G2 and all
mushroom species indicated a negative association with experimental time
units, suggesting successful degradation of lignocellulose complex from
week 0 to week 18. The strength of the time series associations varied
between the measurement parameters, ADF, NDF and ADL. The maximum
degradation over time was reported with mean ADF and mean NDF (R
\textless{} - 0.8; Figure 6 and Figure 7). In contrast, weaker
relationships were recorded in mean ADL across the experimental time
units, where R values ranged from -0.63 to -0.76 (Figure 8). Total
reductions (\% total DM) of ADF, NDF and ADL of the total dry matter are
shown in Tables 8 to 10.

\textbf{Table 8.} Total ADF reduction percentage of substrate after 18
weeks of solid-state fermentation using M1: \emph{P} .
\emph{ostreatus,}M2: \emph{G. lucidum} and M3: \emph{Ganoderma} \#1.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Isolate name} & \textbf{0 weeks \% DM} & \textbf{18 weeks \% DM}
& \textbf{Reduction in total ADF \%}\tabularnewline
\midrule
\endhead
M1 & 64.35\% & 53.53\% & 16.82\%\tabularnewline
M2 & 64.19\% & 52.33\% & 18.48\%\tabularnewline
M3 & 64.12\% & 51.89\% & 19.07\%\tabularnewline
\bottomrule
\end{longtable}

DM: Dry matter, ADF: Acid detergent fibre.

\textbf{Table 9.} Total NDF reduction percentage of substrate after 18
weeks of solid-state fermentation using M1: \emph{P} .
\emph{ostreatus,}M2: \emph{G. lucidum} and M3: \emph{Ganoderma} \#1.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Isolate name} & \textbf{0 weeks \% DM} & \textbf{18 weeks \% DM}
& \textbf{Reduction in total NDF \%}\tabularnewline
\midrule
\endhead
M1 & 78.84\% & 68.63\% & 12.96\%\tabularnewline
M2 & 79.32\% & 62.38\% & 21.36\%\tabularnewline
M3 & 80.04\% & 62.69\% & 21.68\%\tabularnewline
\bottomrule
\end{longtable}

DM: Dry matter, NDF: Neutral detergent fibre.

\textbf{Table 10.} Total ADL reduction percentage of substrate after 18
weeks of solid-state fermentation M1: \emph{P} . \emph{ostreatus,}
M2:\emph{G. lucidum} and M3: \emph{Ganoderma} \#1.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Isolate name} & \textbf{0 weeks \% DM} & \textbf{18 weeks \% DM}
& \textbf{Reduction in total ADL \%}\tabularnewline
\midrule
\endhead
M1 & 22.25\% & 17.71\% & 20.05\%\tabularnewline
M2 & 21.10\% & 17.44\% & 17.60 \%\tabularnewline
M3 & 22.49\% & 16.47\% & 27.10\%\tabularnewline
\bottomrule
\end{longtable}

DM: Dry matter, ADL: Acid detergent lignin.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.} Linear regression scatterplots indicating mean acid
detergent fibre (ADF) degradation over 18 weeks for \emph{G. lucidum}
and \emph{P. ostreatus} G1-G3 substrate grind consistencies\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 7.} Linear regression scatterplots indicating mean
neutral detergent fibre (NDF) degradation over 18 weeks for \emph{G.
lucidum} and \emph{P. ostreatus} G1-G3 substrate grind consistencies.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

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

\subsection*{Discussion}

{\label{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. \emph{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 \emph{P. ostreatus}
and\emph{G. lucidum} (p \textgreater{} 0.05) although the benchmark
strains did reduce observed parameters significantly more than then the
control (p \textless{} 0.05). Therefore, only one grind consistency, G2,
was selected for substrate when comparing the \emph{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 \emph{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
\emph{Ganoderma}\#1 in the repeat of the experiment (measuring only T0
and T18 and using G2 grind consistency) it was determined that
\emph{Ganoderma} \#1 degraded the observed parameters significantly more
than the control and the benchmark strains \emph{G. lucidum} and
\emph{P. ostreatus} (p \textless{} 005).

There were no significant differences between the benchmark
mushrooms\emph{G. lucidum} and \emph{P. ostreatus} tested in their
ability to degrade NDF, ADF or ADL. \emph{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 \emph{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 \emph{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.

\emph{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. \emph{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 \emph{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
of\emph{Ganoderma} 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 initially\textsuperscript{28}. 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{]}. \emph{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. \emph{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 \emph{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 \emph{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. \emph{Pleurotus ostreatus} showed promising
degradation effects, although not as pronounced as the
\emph{Ganoderma}species, could yield a more increased permeability
during rumen digestion due to increased protein production.
\emph{Ganoderma lucidum}displayed a middle of the field result not
digesting as much as\emph{Ganoderma} \#1 although slightly more than
\emph{P. ostreatus} . The NDF portion of the substrate was left more
intact that that of the\emph{Ganoderma} species and with NDF being an
important parameter for efficient digestion by ruminant
microbes\textsuperscript{6} the \emph{P. ostreatus} could yield improved
fermentability.

The success of the SSF using mushrooms could only be verified by actual
digestion by ruminant bacteria either \emph{in vivo} or \emph{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.

\textbf{Acknowledgements~}

No notable Acknowledgments

\textbf{Funding:}

This research was funded by a grant supported by TIA

\textbf{Data Availability}

The data that support the findings of this study are openly available in
OSF at https://osf.io/43fwp/.

\textbf{Conflict of Interest}

The authors confirm that there is, to the best of their knowledge, no
conflict of interest concerning the submission and publication of this
manuscript.

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