Application of solid‐state fermentation using mushrooms for the production of animal feed

The increasing growth of agroindustrial activity resulting in excessive amounts of agriwaste has led to the accumulation of a large quantity of lignocellulosic residues all over the world, in particular in deforestation initiatives for the removal of invasive trees in South Africa. These lignocellulosic residues are rich in energy resources and consist 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, which in turn could yield a new method of producing energy‐rich fodder for ruminant animals. 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, as the outcomes for obtaining potentially high‐quality feeds for nonruminant and ruminant animals are different. The current study presents data on the bioconversion of lignocellulosic substrate using solid‐state fermentation with edible and medicinal mushrooms, Ganoderma lucidumand 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 optimized and the resulting product was analyzed 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 < 0.05) reduction in the acid detergent fiber, acid detergent lignin, and neutral detergent fiber fractions of the biomass, with up to a 20% reduction in indigestible components. This increase in digestibility could contribute to increased energy availability for ruminant animals.

nutritional value to be applied as ruminant animal feed.The solid-state fermentation process was optimized and the resulting product was analyzed 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 < 0.05) reduction in the acid detergent fiber, acid detergent lignin, and neutral detergent fiber fractions of the biomass, with up to a 20% reduction in indigestible components.This increase in digestibility could contribute to increased energy availability for ruminant animals.

| INTRODUCTION
Growth and expansion in the agricultural and food sectors result in the generation and accumulation of a vast amount of agroindustrial waste every year.Agroindustrial 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 et al. [1].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 (SSF) to degrade lignin components in biomass [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 agroindustrial/forestry by-products with low or no economic value to edible biomass [3].
Any biotechnological method in which living organisms are cultivated on nonsoluble or solid material in the absence (or near absence of free water) is recognized as SSF [4].SSF 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 utilization during rumen digestion [6].Basidiomycetous white-rot fungi, such as Pleurotus ostreatus, digest lignocellulose in anticipation of producing fruiting structures (mushrooms) and display a unique strategy to colonize 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 toward 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 the 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 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 solution.To increase sustainability, Lukomska et al. [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 an SSF method for the selective delignification of A. mellifera using basidiomycetous fungi, that is, mushrooms, as biological agents.
F I G U R E 1 A large Acacia mellifera tree with a surrounding impenetrable thicket of seedlings (picture by author).

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

| Substrate for SSF
A. mellifera was chosen as the preferred substrate due to the excessive volumes available in central South Africa.The 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, <1 cm).The woody substrate was allowed to "rest" for 2 weeks before use to circumvent any passive host immunity to the mushroom strains.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.

| Substrate cultivation bags
The substrate was soaked in demineralized water for 24 h for hydration.The hydrated substrate was subsequently drained until no free running water was observed.The substrate moisture content was standardized at 75% moisture.The substrate was packed into polypropylene bags with 0.45 µm pore size filter patches to contain a total of 400 g of hydrated A. mellifera wood chips in the three different size categories, that is, all substrate bags were autoclaved for 90 min at 121°C.Over the course of 20 weeks, 180 cultivation bags were used in duplicate, with sample bags selected at random every 2 weeks for analysis.

| Mushroom cultures
The purchased mushroom cultures were received on Difco™ potato dextrose agar (PDA) slants.Upon reception, the cultures were transferred aseptically to fresh F I G U R E 2 Constituents of the carbohydrate fraction of woody plant residues and the inherent fermentability of the basic building blocks.ADF, acid detergent fiber; NDF, neutral detergent fiber.
PDA plates and allowed to grow for 7 days before being used as spawn inoculum.All species applied were previously characterized on a genomic level using the internal transcribed spacer (ITS gene region).

| Mushrooms spawn production
Sorghum grains were soaked for 24 h for hydration.The grains were removed from the soaking water and surfacedried by rolling on a towel until no longer visibly wet on the surface.Moisture contents were standardized at 70%.The grains were then placed in polypropylene cultivation bags with air and 0.45 µm pore size filters and sterilized by autoclaving for 90 min at 121°C.After being cooled to room temperature, the bags were inoculated by adding a 1 × 1 cm square of PDA agar covered with mushroom mycelium.Bags were then incubated at 25°C until fully colonized.

| Substrate inoculation
Each 400 g substrate bag was inoculated with 40 g of sorghum grain mushroom spawn and shaken to distribute the inoculum.The substrate bags were placed on shelves in a growth room at 25°C and 85% humidity (Figure 3).The growth room was kept dark to stimulate mycelium growth.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.

| Fiber content analysis
Samples were analyzed and calculations were performed for neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) using the ANKOM 220 Fiber Analyzer (ANKOM Technology Corporation) according to the van Soest et al. method [12].NDFom-NDF was not assayed with a heat-stable amylase and was expressed exclusive of residual ash.ADFom-ADF was expressed exclusive of residual ash.Lignin (sa)-Lignin was determined by the solubilization of cellulose with sulfuric acid [13].
The ADF content of samples was calculated as follows:

| 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 analyzed by combustion assay [14].A 1-g sample was weighed accurately to determine the CP content by inserting it into a Leco Nitrogen analyzer and the total N content was determined by combustion in oxygen [15].A factor of 6.25 was used to convert the N content of the samples to CP content [14].

| Gross energy determination
One gram of the sample was analyzed in triplicate to determine the GE content according to the procedures described by the Association of Official Analytical Chemists [16].
F I G U R E 3 Substrate bags colonized by Ganoderma lucidum 7 days after inoculation.

| Factorial design and data analysis
Data processing and analyses were performed with R version 4.0.2[17] within R Studio version 1.2.5042 [18].Data were imported using the "gsheet" package [19].Data exploration, wrangling, and visualization were conducted using the "tidyverse" package [20].Analysis of variance (ANOVA; α = 0.05) of the data was performed using base R functions.ANOVA was applied to determine if mushroom species (G.lucidum, Ganoderma #1, and P. ostreatus) and grind (sieve sizes of 2-5 cm, 1-2 cm, and <1 cm, for grind 3, 2, and 1, respectively) affected dependent variables (ADF, ADL, and NDF) from the first and last sampling period, 0-18 weeks.Means were separated using the least significant difference (LSD) test function from the "agricolae" package [20].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's correlation coefficient was applied to the scatter plots and plotted with the "ggpubr" package [21] to determine the strength of the associations presented.

| Quantifying SSF
ANOVA indicated time of experimental sampling as the only main effect resulting in significant differences (p < 0.05) for the mean ADF (Table 1) and mean NDF (Table 2).However, mushroom species and the time of experimental sampling contributed to significant differences in the mean ADL (Table 3).Means separation indicated all Time 0 samples held the highest concentration for all measured degradation parameters, ADF, NDF, and ADL.After 18 weeks of SSF, all concentrations had collectively reduced by 11.66%, 14.84%, and 4.76% (equating to g/100 g DM) for mean ADF, NDF, and ADL, respectively (Table 4).The mushroom species that had the supreme ability to degrade ADL was Ganoderma #1, where the lowest mean ADL (16.47 g/100 g DM) was recorded.Consequently, P. ostreatus and G. lucidum had reduced abilities to degrade ADL compared to that of F I G U R E 4 Crude protein (CP) content (g/kg dry matter [DM]) of the substrate after 18 weeks of solid-state fermentation using Ganoderma lucidum, Pleurotus ostreatus, and Ganoderma #1 compared to an undigested control.
Ganoderma #1, although all mushroom species held the ability to significantly degrade ADL through SSF collectively (Table 4).When viewed individually (Table 5), as indicated by means separation, Ganoderma #1 showed increased degradation of all measured parameters, with the exception of ADF.Additionally, Ganoderma #1 performed significantly greater in degradation capacity when compared to benchmark strains G. lucidum and P. ostreatus (p < 0.05), which did not F I G U R E 5 Gross energy (GE) content (MJ/kg dry matter [DM]) of the substrate after 18 weeks of solid-state fermentation using Ganoderma lucidum, Pleurotus ostreatus, and Ganoderma #1 compared to an undigested control.
T A B L E 1 Analysis of variance for mean ADF across three mushroom species (Ganoderma lucidum, Ganoderma #1, and Pleurotus ostreatus), with three substrate grind consistency G2, where samples were taken from experiment initiation (Week 0) to competition (Week 18).degrade parameters significantly when compared with each other, although they did degrade parameters significantly more than the control (p < 0.05).

| Total fiber reduction
To determine the degradation of the lignocellulose complex for the duration of the experiment, linear regression scatterplots and Pearson's correlation coefficients (R) were conducted.All Pearson's correlation coefficients, with the exception of the mean ADL percentage at grind 1 of G. lucidum (p ~0.053), were significant (α = 0.05).SSF across grind consistency G2 and all mushroom species indicated a negative association with experimental time units, suggesting successful degradation of lignocellulose complex from Weeks 0 to 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 < −0.8; Figures 6 and 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 DM are shown in Tables 6-8.

| 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 colonization of substrate bags was achieved with all strains completing colonization within 7 days after inoculation.There were no significant differences observed in ADF, ADL, or NDF reduction between the benchmark isolates P. ostreatus and G. lucidum (p > 0.05), although the benchmark strains did reduce observed parameters significantly more than the control (p < 0.05).Therefore, only one grind consistency, G2, was selected for substrate when comparing Ganoderma #1.It was interesting that no differences were observed as particle size was considered a key factor in studies performed by Batool et al. [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 colonization of the fungus, but by decreasing the particle size too much, the substrate became anaerobic, which diminished 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 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 Ganoderma #1 in the repeat of the experiment (measuring only Time 0 and Time 18 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 < 0.05).There were no significant differences between the benchmark mushrooms G. lucidum and P. ostreatus, which were tested for 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 significant degradation of parameters 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 (Tables 6-8), the effects can be visualized 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 increased accessibility to cellulose.When considering that not only degradation but also modification took place, where lignin strands were not necessarily broken down but T A B L E 5 Means separation through Fisher's LSD for mean ADF, NDF, and ADL of substrates M1 (Pleurotus ostreatus), M2 (Ganoderma lucidum), M3 (Ganoderma #1), and S (untreated Acacia mellifera) between all fungal treatments expressed as percentage (%) of DM.

Treatment
Mean 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 seen 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 GE available was only reduced by roughly 2%, while CP increased by almost 30%.It has been shown that an enhancement in protein can be achieved by the treatment F I G U R E 6 Linear regression scatterplots indicating mean acid detergent fiber degradation over 18 weeks for Ganoderma lucidum and Pleurotus ostreatus G1-G3 substrate grind consistencies. of fungi such as mushrooms [26].CP 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 in protein was not the primary goal of the experiment.Interestingly, although 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.
A. mellifera contains a GE of 15.8 MJ/kg DM.This has 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 more available energy for ruminant digestion.P. ostreatus delivered a less significant delignification but yielded a significantly higher CP increase of 3.5%-6.3%.The inherent problem with using P. ostreatus for the SSF purpose of this trial was the lack of overcoming the tree's 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 initially cleaving the extended strands [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.However, this can be seen as large variations in the data analysis of the ADL fraction when compared to the confidence intervals of NDF or ADF fractions.Where stronger relationships would yield narrower confidence intervals as the variables in the model would account for a greater prediction of the population mean.
SSF is a valuable tool for the biotechnological sector and allows for numerous possibilities in the production of useful products from otherwise unusable wastes [16].A. mellifera is abundant in central to northern South Africa grasslands/ veld/and so on, 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 pose several problems for farmers, particularly as the primary means of disposal is the burning of biomass [11].South Africa has in recent years experienced severe drought, which prompted the need for alternative sources of feed for domesticated livestock.A. 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 it did serve as a short-term solution to starving animals.A practical solution would be to develop a pretreatment 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 being able to withstand competition from cosmopolitan contaminant microbes and host plant defenses.The need for a South African isolate was necessary to avoid possible contamination of South African biodiversity.The Ganoderma #1 isolate proved to be remarkable in both its lignolytic abilities, and energy efficiency, that is, being able to digest significant amounts of lignin while leaving the desired cellulose fraction intact, and without utilizing 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 the 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.Even though the degrading effects of P. ostreatus were not as strong as those of 423 Ganoderma species, they were still promising and could lead to increased permeability during 424 rumen digestion due to more protein production.G. lucidum displayed a middle-of-the-field result, not digesting as much as Ganoderma #1, although slightly more than P. ostreatus.The NDF portion of the substrate was left more intact than that of the Ganoderma species, and with NDF being an important parameter for efficient digestion by ruminant microbes [6], 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 digestibility 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 [29].

ACKNOWLEDGMENTS
This research was funded by a grant supported by TIA.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.| 1163 ADF (g/kg DM) = Sample weight after boiling (g DM) − Ash weight (g DM) /ADF (g/kg DM) /Weight of sample (g DM) × 1000.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) × 1000.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) × 1000.

F I G U R E 7
Linear regression scatterplots indicating mean neutral detergent fiber degradation over 18 weeks for Ganoderma lucidum and Pleurotus ostreatus G1-G3 substrate grind consistencies.host defense as it can only survive saprophytically on decaying host plants instead of the parasitic abilities of Ganoderma spp.

F I G U R E 8
Linear regression scatterplots indicating mean acid detergent lignin degradation over 18 weeks for Ganoderma lucidum and Pleurotus ostreatus G1-G3 substrate grind consistencies.

T A B L E 6
Total ADF reduction percentage of the substrate after 18 weeks of solid-state fermentation using M1: Pleurotus ostreatus, M2: Ganoderma lucidum, and M3: Ganoderma #1.