Statement of Novelty

This study firstly investigated naked oat straw pretreated by white rot fungi to improve its nutritional value as ruminants feed. The production and cultivation areas of naked oat are expanding owing to the health benefits obtained by its flour. Hence, it was necessary to develop a useful and proper method to make its by-product valorization. While, seldom data could obtain on the biological pretreatment of naked oat straw to improve its utilization. In present study, white rot fungi of Irpex lacteus can not only degrade acid detergent lignin but also alter lignocellulosic structure then improve the nutritional value of naked oat straw.

Introduction

Crop straw, which is composed of cellulose, hemicellulose, lignin, and a small amount of crude protein, is an abundant global resource that can potentially be used to feed ruminants [1, 2]. However, the presence of the recalcitrant lignin matrix, which forms a tight vascular structure that prevents rumen microorganisms or enzymes from utilizing accessible holocellulose polymers, leads to low digestibility and poor palatability thereby discouraging its use as fodder for ruminants [3]. As a result, a number of studies have investigated pretreatment strategies to break down this matrix and thus increase the potential for ruminant consumption [4]. In particular, the pretreatment of crop straw using strategies that decompose the lignocellulosic structure to release holocellulose and make it accessible for microorganisms has received recent focus [5, 6].

Naked oat (Avena nuda L.) is an annual herbaceous species originating from China that differs from hull oat (Avena sativa L.) in terms of its morphology and nutritional value. Its flour consists of various nutritional substances and trace elements like calcium, phosphorus, and iron that are essential to the health and that can prevent some cardiovascular diseases [7]. According to statistics published by the Food and Agriculture Organization in 2017, the annual production of naked oats in China was 0.9 million tons. Given a residue-to-product ratio of 1.5, this equates to a yield of naked oat straw of approximately 1.4 million tons. With increasing awareness of the importance of health among consumers, the cultivation area and production of naked oats are both expanding. Increased cultivation will produce a large volume of by-product that may put pressure on the environment. Hence, useful approaches are required to improve the efficient utilization of crop straw.

Recently, biological pretreatment with white rot fungi has attracted attention because it only requires mild processing, has excellent effects, and is environmentally friendly compared to physical, chemical, or combined pretreatment strategies [2, 8]. White rot fungi, which is saprophytic on wood, can mineralize lignin into CO2 and H2O in nature [9, 10]. The lignin degradation ability of individual fungus species depends not only on their intrinsic characteristics but also on the type of biomass [11, 12], and degradation performance for the same biomass differs among species and strains [13]. In our previous study, we inoculated wheat straw and corn stover with seven species of white rot fungi; of these species, Irpex lacteus performed well, not only altering the multi-scale structure of straw but also improving rumen fermentation in vitro [14, 15]. However, even though a large volume of research has analyzed the pretreatment of a diverse range of biomass types with various fungus species, the effect of white rot fungus pretreatment on naked oat straw for use as a ruminant feed has not yet been investigated.

In the present study, four white rot fungus species—Phanerochaete chrysosporium, Pleurotus ostreatus, I. lacteus, and Phlebia acerina—were used to treat naked oat straw for solid-state fermentation. Changes in the chemical composition were analyzed, morphology and structure of oat straw were investigated using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), followed by an analysis of in vitro ruminal digestion to determine the nutritional value of the straw after pretreatment. These characteristics were used to determine the performance of the four fungus species in terms of decomposing the lignocellulosic structure and improving in vitro ruminal digestion.

Materials and Methods

Fungal Preparation

Phanerochaete chrysosporium CGMCC 5.829, Pleurotus ostreatus CGMCC 5.374, Irpex lacteus CGMCC 5.809 and Phlebia acerina CGMCC 5.927 were procured from the China General Microbiological Culture Collection Center (CGMCC) in Beijing. The fungi were preserved on potato dextrose agar (PDA) culture tube slants at 4 °C. The initial cultures were grown on PDA plates at 28 °C until mycelia covered the entire agar plate. The plates were then stored at 4 °C for subsequent inoculation.

Substrate Preparation and Inoculation

Naked oat was harvested in July 2018 in Inner Mongolia, China. The straw was air-dried in the field then transported back to the laboratory. The initial moisture of the straw was 4.27%. It was chopped and ground with an RT-34 hammer mill (Rong Tsong Precision Technology Co., Taiwan) then passed through a 20 mesh screen. The raw straw was primarily composed of cellulose (CL; 336.6 g/kg dry matter [DM]), hemicellulose (HC; 288.9 g/kg DM), acid detergent lignin (ADL; 54.4 g/kg DM), and crude protein (CP; 42.2 g/kg DM).

The screened straw was weighed in a valve bag, and distilled water was added to adjust the moisture to 70%. After thorough mixing by hand, the straw was stored in the refrigerator at 4 °C for 3 h to allow the water to fully infiltrate the straw. The wet substrate was then distributed between glass petri dishes (about 10 g per dish), labeled, and weighed. The petri dishes containing the wet straw were sterilized at 121 °C for 20 min then cooled in an aseptic room for subsequent inoculation.

The sterilized wet straw was inoculated with the four fungus species using perforators with a diameter of 0.5 mm. Each of the culture dishes was inoculated with three agar plates fully colonized with hyphae. The control was inoculated with the same amount of PDA with no fungi. All of the dishes were placed in a mold incubator at 28 °C, with a relative humidity of 80%. Every week, three random dishes for each treatment were removed then dried in a blast-drying oven at 65 °C for 24 h to a constant weight for subsequent chemical composition and nutritional value analysis. Three replicates were used for all experimental assays.

Chemical Analysis

The weekly oven-dried samples were analyzed to determine DM content and calculate the loss of DM. Based on a modified version of Van Soest’s method [16], the contents of neutral detergent fiber (aNDF), acid detergent fiber (ADF) and ADL were calculated. Cellulose content was calculated as the difference between the ADF and ADL, while hemicellulose content was calculated as the difference between the aNDF and ADF. The neutral detergent solute (NDS) refers to the portion of the straw extracted by a neutral detergent and was calculated by subtracting the aNDF from 1000. The CP content was determined by measuring the nitrogen content using the Kjeldahl method (ISO 5983-1,2005) and the result was then multiplied by 6.25.

Scanning Electron Microscopy (SEM)

The microscopic morphological structure of the straw was observed using SEM (S-3400N, Hitachi, Tokyo, Japan). The samples were coated with a thin layer of gold using a sputter coater, and SEM images were then obtained under an accelerating voltage of 10 kV and a magnification of × 1000.

Fourier Transform Infrared Spectroscopy (FTIR)

The functional groups in the straw were investigated using FTIR. Samples were dried at 105 °C for 4 h and then stored in an airtight desiccator until further testing. Dried potassium bromide (spectral level) was mixed with the dried samples at a ratio of 100:1, which were then fully ground and pressed into tablets using a YP-2 tableting machine (Shanghai Shan Yue Science Instrument Co., Ltd.). Sample spectra were obtained using an average of 32 scans over a range of between 500 and 4000 cm−1 with a spectral resolution of 2 cm−1 per sample using a PerkinElmer Spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, MA, USA). Baseline and ATR correction for variation in penetration depth and frequency were conducted using the spectrum software supplied with the equipment.

In Vitro Ruminal Digestion

In vitro ruminal digestion was used to evaluate the effect of the fungal treatment on the nutritional value of the straw. In brief, fresh rumen fluid was collected from four rumen-fistulated Angus bullocks fed with a corn silage and wheat straw diet before morning feeding. The rumen fluid was mixed with a buffer solution at a 1:2 (v/v) ratio under continuous flushing with CO2. The buffer solution was formulated following the process outlined by Menke [17].

ANKOM F57 filter bags filled with the samples and the blank control were submerged in the buffer solution in jars, placed in a DaisyII Incubator (ANKOM TECHNOLOGY), and allowed to ferment at 39 °C. After 48 h, the jars were placed in iced water to immediately terminate the reaction. The filter bags were removed, washed with running tap water, and then rinsed with distilled water. Clean tissue paper was used to extract the excess water, and the samples were then dried in a blast-drying oven at 65 °C to reach a constant weight for the calculation of in vitro dry matter digestion (IVDMD). The digestion of aNDF, ADF, cellulose, and hemicellulose after 48 h was also calculated.

In Vitro Dry Matter Digestion (IVDMD)

The IVDMD was calculated using the following equation:

$$DMD\left( \% \right) = \frac{{m_{1} - m_{2} }}{{m_{1} }} \times 100$$

where DMD denotes the dry matter digestion, m1 is the weight before digestion (g), and m2 is the weight after digestion (g).

In Vitro N Digestion (IVND)

The in vitro N digestion (IVND) was calculated using the following equation:

$$ND\left( \% \right) = \frac{{m_{1} \times c_{1} - m_{2} \times c_{2} }}{{m_{1} \times c_{1} }} \times 100$$

where ND represents the digestion of aNDF, ADF, CL or HC, m1 is the weight before digestion (g), m2 is the weight after digestion (g), c1 is the sample content of the corresponding substance before digestion (g/g), and c2 is the sample content of the corresponding substance after digestion (g/g).

Statistical Analysis

The effect of the four fungus species on the chemical composition of naked oat straw and in vitro ruminal digestion was analyzed using one-way analysis of variance (ANOVA) by SPSS, version 22.0 (IBM Corp., Armonk, NY, USA). Two-factor ANOVA was used to analyze the fungal (F) and treatment days (D) and the interaction of the two (F × D). Significance was determined by a P-value lower than 0.05.

Results and Discussion

The four fungus species all grew well on naked oat straw during the pretreatment stage, with no mold visually observed in any of the treatments. While, the speed of fungi growth varied owing to their own intrinsic characteristics, P. chrysosporium and I. lacteus fully colonized on the substrate in 5 and 6 days, respectively, P. ostreatus and P. acerina needed 7 and 8 days. The healthy growth of a fungi on the substrate is a prerequisite for the fermentation process to occur. The rapid growth of mycelia allows inoculated fungi to become dominant on the substrate, preventing undesirable molds or bacteria from contaminating the substrate [18].

Change in Chemical Composition

The contents of aNDF and ADF in all treatments were significantly (p < 0.001) higher in the first week of fermentation and then declined continuously afterward, except for P. chrysosporium, which decreased at the beginning of treatment. By the end of pretreatment with P. chrysosporium, the aNDF and ADF contents had decreased 20.99% and 21.74% respectively (Table 1). After 2 weeks of treatment with P. ostreatus, the ADF content basically stabilized, in contrast to the other pretreatments. The ADL content increased slightly in the first week treatment then declined gradually in all treatments which was also founded in previous reports [14, 19, 20]. It was speculated that more accessible carbohydrates were originally the primary source for fungal growth at this time, thus the relative levels of ADL were elevated. After whole pretreatments, the ADL degradation was highest with the use of P. acerina (from 72.7 g/kg DM to 55.8 g/kg DM; p < 0.001) followed by P. chrysosporium (63.4 g/kg DM; p < 0.001). Correspondingly, ADL degradation by P. ostreatus and I. lacteus was similar and a little weaker than that of the other two species. The NDS, which includes fat, starch, water-soluble carbohydrates, and protein, represents the ideal nutritional component for rumen microorganisms [21]. After pretreatment by I. lacteus and P. acerina, the NDS levels increased because the depolymerization of the lignin structure allowed nutritional compounds to accumulate. It also indicated that holocellulose was not completely degraded into carbon dioxide and water but was instead partly converted into low-molecular-weight carbohydrates [21]. The CP contents in the treated straw were all slightly higher than in the sterilized straw, with samples pretreated with P. chrysosporium increasing the most, followed by I. lacteus (p < 0.001). At the end of the treatment with P. chrysosporium, CP contents increased to 58.5 g/kg DM compared to 42.2 g/kg DM for the control. Even though CP levels were low in the raw straw, after fungal pretreatment, CP levels increased compared to the control. Previous reports on the use of different fungus species to solubilize the CP in wheat straw have shown that fungus treatment was helpful in increasing CP levels [22]. This represents a potential advantage for biological pretreatment compared with other methods, and provides new insight into the use of biological pretreatment to produce ruminant fodder. However, further investigation is required to elucidate the detailed mechanisms underlying the changes in CP levels in response to fungal treatment.

Table 1 Chemical composition (g/kg DM) of naked oat straw after 0, 7, 14, 21 and 28 days of treatment with different fungi
Full size table

The DM continuously decreased for all the treatments. By the end of treatment, P. chrysosporium had caused a DM loss of 34.82%, while the DM loss caused by the other species were all below 20%, with P. ostreatus treatment having the smallest loss at 13.59% (Table 2). The loss of DM in the pretreated straw increased over time due to the fungi using carbohydrates for its own growth. The loss of DM naturally differs between different fungus species, but it is also affected by biomass type. At the end of the treatment, P. chrysosporium caused a DM loss of 34.82% in the present study, which was lower than the 45.17% and 50.1% previously reported for P. chrysosporium on wheat straw and corn stover, respectively [14, 15]. The DM loss caused by P. ostreatus, I. lacteus and P. acerina was 13.59%, 18.33%, and 18.15%, respectively. At the end of treatment, ADL loss caused by P. acerina, P. chrysosporium, I. lacteus, and P. ostreatus was 46.51%, 44.20%, 36.54%, and 31.50%, respectively. The cellulose degradation caused by P. chrysosporium was 49.70%, much higher than the other treatments, which were all below 20%, with P. ostreatus only degrading cellulose by 5.04%. All of the treatments caused hemicellulose degradation in the order of P. chrysosporium > P. acerina > I. lacteus > P. ostreatus. Overall, the most significant change in chemical composition was caused by P. chrysosporium treatment, and the least significant by P. ostreatus. In general, white rot fungi can be roughly divided into two categories: selective or non-selective. The selectivity value (SV), which is the ratio of lignin degradation to cellulose loss, is an important factor in evaluating the potential of fungi for degrading lignin and retaining cellulose [23]. As a traditional model fungus, P. chrysosporium has attracted significant attention due to its powerful ability to degrade lignin. However, this decomposition of lignin is typically accompanied by severe cellulose loss, thus it is considered to be a non-selective fungus. This non-selectivity of P. chrysosporium was confirmed in the present study. It was also found that the NDF degradation at the beginning of treatment was the result of non-selective degradation by P. chrysosporium. The other three fungus species all exhibited high SVs, with P. ostreatus having the highest.

Table 2 Loss (%) of each component of naked oat straw after 7, 14, 21 and 28 days of treatment with different fungi
Full size table

Morphological Structure

The micro-morphological structure of the straw is illustrated in the SEM images in Fig. 1. The sterilized straw exhibited a tight vascular structure and the surface was quite smooth and compact, while the surface of the pretreated straw was partially destroyed by fungi but to different degrees. It was evident that the highly dense vascular structure loosened and became more porous after pretreatment. In addition to P. ostreatus treatment, there were a large number of pits and holes on the surface of the straw at the end of the pretreatment process. The same situations were also discovered in other reports that have investigated the diverse mechanisms involved in structural decomposition by different fungi [14]. Meanwhile, the compact vascular structure loosens and becomes more porous, it is easier for rumen microbes to access nutritional substances, thus improving in vitro ruminal digestion [24]. The reduction in the strength of the chemical bonds observed in the FTIR spectra in the present study thus confirmed that the lignocellulose structure was decomposed by the fungi. The functional groups in the naked oat straw were investigated using FTIR spectra (Fig. 2). Characteristic peaks of the related functional groups were used to determine the structure of the lignocellulosic biomass (Table 3). The intensity of the absorbance in the straw pretreated with fungi was lower compared to the sterilized straw, especially for the main peaks at 1055 cm−1, 1249 cm−1, 2918 cm−1, and 3400 cm−1, illustrating the effectiveness of fungus pretreatment. The absorption peak at approximately 1603 cm−1 was ascribed to the aromatic ring C=C and C–O stretching in lignin, and the lower intensity suggests the partial removal or degradation of lignin. The degree of the loss of intensity roughly corresponds to the changes in the chemical composition. The chemical bonds at 1249 cm−1 associated with C–O stretching between hemicellulose and lignin were also affected, indicating that lignin degradation was accompanied by the decomposition of cellulose and hemicellulose [25].

Fig. 1
figure 1

SEM analysis of substrates: a sterilized substrate, b pretreatment by Irpex lacteus, c pretreatment by Pleurotus ostreatus, d pretreatment by Phlebia acerina, and e pretreatment by Phanerochaete chrysosporium

Full size image
Fig. 2
figure 2

The FTIR spectra of naked oat straw pretreated by different fungus species. Control (green), Pleurotus ostreatus (black), Phanerochaete chrysosporium (red), Irpex lacteus (purple), Phlebia acerina (blue)

Full size image
Table 3 The correspondence between wavenumbers and functional groups
Full size table

In Vitro Ruminal Digestion

Results for the in vitro ruminal digestion of the pretreated and sterilized straw are presented in Table 4. The DMD and CLD of the sterilized straw were 395.1 g/kg and 276.7 g/kg, respectively. The DMD was improved by all fungus species except for P. chrysosporium, which had a DMD of 373.3 g/kg, lower than the sterilized straw. While, the NDFD, ADFD, CLD, and HCD were all higher in the pretreated straw compared to the sterilized straw. The serious loss of DM and reduction in NDS content caused by P. chrysosporium may have weakened the nutritional value compared to control and lowered the digestibility. However, due to high levels of lignin decomposition, the NDFD, ADFD, CLD, and HCD of straw pretreated with P. chrysosporium were all higher compared to the sterilized straw. This is because the ADL degradation exposed the holocellulose and increased the contact with rumen microorganisms, leading to relative digestion improvement. The same results have been reported by previous research. For example, the in vitro gas production of biomass pretreated with P. chrysosporium decreased compared to control [14, 26]. Therefore, even though P. chrysosporium has excellent ADL degradation ability, it cannot improve the nutritional value of the biomass after fermentation, indicating that it is not suitable for use in the pretreatment of naked oat straw.

Table 4 In vitro digestibility (g/kg) of chemical constituents of naked oat straw in different treatment groups
Full size table

Pleurotus ostreatus, an edible mushroom, exhibits a high SV in the degradation of lignocellulosic biomass, with a mechanism that differs compared to other fungi. In this study, the ADL degradation caused by P. ostreatus was moderate, but NDS levels decreased slightly after pretreatment. Correspondingly, a slight improvement was observed in the digestion of straw pretreated with P. ostreatus compared to the control. The SEM images also showed that there were no micropores observed on the surface of the pretreated straw, which would not facilitate in vitro ruminal digestion. Indeed, it has been reported that the in vitro gas production of different types of biomass pretreated with P. ostreatus increased in some studies but decreased in others [4, 14, 20], and this depends not only on the biomass type but also the strains of the same species.

Irpex lacteus is a representative fungus that has been used in traditional Chinese medicine for the treatment of chronic glomerulonephritis treatment and the lowering of blood pressure [27]. Previous studies that have employed I. lacteus to pretreat different types of biomass reported significant improvement in in vitro gas production and the rate of enzymatic hydrolysis [28, 29]. These results are consistent with the changes in the composition of the straw in the present study. In the straw pretreated with I. lacteus, the structure was altered, ADL was decomposed, and NDS levels were moderately elevated. In particular, in vitro digestion was significantly higher (p < 0.001) compared with the other treatments. Past studies have also highlighted this species as an ideal fungal agent for use in biological treatment.

Phlebia acerina showed a strong ADL degradation ability in present study. Some researchers have inoculated paddy straw with Phlebia species and demonstrated improved in vitro digestibility [30]. In contrast, the digestibility of naked oat straw pretreated with P. acerina was lower than that pretreated with I. lacteus and was not appreciably higher than the control. This conflicted with the fact that this species demonstrated the highest ADL degradation and elevated NDS levels. It has been speculated that this fungus produces compounds that inhibit fermentation by rumen microorganisms, resulting in in vitro digestibility that is not significantly improved [31]. Further investigations should be conducted to identify the compounds this fungus secretes and their effect on rumen microorganisms.

In summary, the improvement of in vitro ruminal digestion is result from the interaction of a diverse range of factors. Due to the decomposition of the lignin matrix and the release of holocellulose, more accessible cellulose is exposed then enhanced contact with rumen microbes. The elevated NDS levels also contribute to the improvement of in vitro digestion. However, inhibitory compounds secreted by the fungi may inhibit ruminal fermentation.

Conclusion

All four fungus species degraded lignocellulose and altered the vascular structure of naked oat straw. In terms of DM loss, cellulose loss, ADL degradation, and structural decomposition, I. lacteus and P. acerina outperformed the other two fungus species. In addition, the elevated CP and NDS levels contributed to the significant improvement of in vitro digestion when the straw was pretreated with I. lacteus. As a result, I. lacteus was the most effective fungus investigated in this study in terms of not only altering the lignocellulosic structure but also improving the nutritional value of naked oat straw.