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Article

Improving the Nutritional Value and Safety of Cotton Stalk Feed via Response Surface Methodology and Co-Fermentation Techniques

by
Kunyi Li
1,
Yuansheng Xu
1,
Kai Guo
1,
Weidong Cui
1,
Yang Li
1,* and
Min Hou
1,2,*
1
Xinjiang Laboratory of Special Environmental Microbiology, Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Saybagh District, Urumqi 830091, China
2
Key Laboratory of Western Arid Region Grassland Resource and Ecology of Ministry of Education, College of Grassland Science, Xinjiang Agricultural University, Saybagh District, Urumqi 830052, China
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(3), 124; https://doi.org/10.3390/fermentation11030124
Submission received: 15 February 2025 / Revised: 25 February 2025 / Accepted: 27 February 2025 / Published: 5 March 2025
(This article belongs to the Section Fermentation Process Design)
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Abstract

Cotton stalks, a major agricultural byproduct, are challenging to decompose naturally; however, they can be transformed into valuable animal feed through microbial fermentation. This study identifies Aspergillus niger HQXY as the most efficient cellulase-producing strain out of six evaluated strains, using it to ferment cotton stalks and significantly degrade cellulose and hemicellulose. By optimizing solid-state fermentation conditions via response surface methodology, the crude fiber content of the cotton stalks was reduced by 34%. A novel sequential co-fermentation approach combining Aspergillus niger with probiotics (Bacillus licheniformis, Candida utilis, and Lactobacillus casei) further enhanced the feed’s nutritional profile. The optimal results were obtained using a 1:1:1 ratio of strains (Aspergillus niger, Bacillus licheniformis, and Lactobacillus casei) at a 8% inoculation rate over 30 days. This co-fermentation strategy lowered the pH and reduced gossypol to 15.5 mg·kg−1. The findings highlight the effectiveness of Aspergillus niger HQXY and probiotics in improving the quality of cotton stalks, by reducing crude fiber and gossypol content, thus offering a promising method for the sustainable utilization of agricultural waste as high-quality animal feed.

1. Introduction

China is the world’s largest cotton producer, accounting for nearly 30% of global annual cotton production and generating approximately 40 million tons of cotton stalk annually [1,2]. Globally, the yearly cotton stalk supply of cotton stalks ranges from 90.3 million tons to 129 million tons, with an increasing trend [3]. However, traditional disposal methods, such as burning and landfilling, cause environmental pollution and degrade soil physicochemical properties, leading to soil degradation and loss of microbial communities [4,5]. Therefore, there is an urgent need for sustainable and environmentally friendly approaches to utilize this abundant agricultural byproduct.
Microbial fermentation technology provides an innovative solution for the resource utilization of cotton stalks by breaking down the lignocellulosic structure and successfully converting agricultural waste into high-value ruminant feed [6]. This technology not only improves the digestibility and nutritional value of cotton stalks but also alleviates the dual pressures of environmental pollution and feed shortage, thereby promoting sustainable development in livestock farming [7,8]. Cotton stalks are rich in crude protein, fat, and nutrients such as nitrogen and phosphorus. However, their dense cellulose–hemicellulose–lignin complex structure forms a natural barrier to degradation, significantly limiting enzymatic hydrolysis efficiency [9,10]. Fermentation using a single strain has notable limitations. For instance, while Trichoderma species possess an efficient cellulase system, their metabolic products are limited. Co-fermentation involves the synergistic action of fungi (e.g., Aspergillus niger) and cellulose-degrading bacteria (e.g., Bacillus subtilis Candida utilis), where fungi preferentially degrade cellulose and hemicellulose, followed by bacterial decomposition of cellulose, releasing carbohydrates, and secretion of proteases to release nitrogen sources, thus enhancing the yield of reducing sugars and protein content [11,12]. Zhu et al. [13] found that the inoculation of cellulose-degrading microbial consortia into sweet sorghum silage significantly improved the cellulose degradation capacity. Perizat S. et al. [14] found that through the use of fungal and probiotic consortia, the protein content of the feed was increased considerably compared to fermentation with a single strain. Co-fermentation technology can reduce feed costs and boost the production efficiency of livestock farming. Simultaneously, by converting agricultural waste into high-value products, environmental pollution is decreased, and new raw material sources for producing bioethanol and other biofuels are provided, offering significant industrial application prospects.
Recent advances in microbial fermentation technology have shown that certain fungi, such as Aspergillus niger, can secrete lignocellulolytic enzymes, including lignin-degrading enzymes and cellulases, which significantly enhance hydrolysis efficiency [15,16]. For instance, the combination of Aspergillus niger with Trichoderma koningii in solid-state fermentation of tea residue has been reported to increase crude protein content and promote fiber degradation [17,18]. Furthermore, Aspergillus niger has been confirmed as safe for use as a feed additive [19]. However, the effectiveness of microbial fermentation is still limited by issues such as insufficient enzymatic activity of single strains, low nutrient release rates, and suboptimal strain combinations. Therefore, selecting appropriate microbial strains and optimizing fermentation conditions are crucial for improving the quality of cotton stalk feed.
In this study, we aimed to identify the most efficient cellulase-producing strain of Aspergillus niger and optimize the solid-state fermentation conditions for cotton stalks. Furthermore, we explored the effects of a novel sequential co-fermentation approach combining Aspergillus niger with probiotics (including Bacillus licheniformis, Candida utilis, and Lactobacillus casei) on the nutritional profile of cotton stalk feed. This research aims to explore innovative methods for utilizing cotton stalks more effectively, enhancing cellulase production, and improving the nutritional value and safety of cotton stalk fermented feed.

2. Materials and Methods

2.1. Materials

The cotton stalk was acquired from Xinjiang Kuler Yuli County Tongfeng Oil and Grease Co. (Xinjiang, China). Cotton stalk was chopped into 2–3 cm with a blender, crushed by a pulverizer (Zhigao, FTT-2500G, Quzhou, China), passed through a 40 mesh sieve, and intercepted on a 100 mesh sieve, so that the diameter of the particles was between 40 and 100 mesh. Six strains of Aspergillus niger, Candida utilize, Bacillus licheniformis, and Lactobacillus casei, were provided from the Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, namely L3, L4, HQXY, HQZD, Z3, Z4, C, B, and L, respectively. These strains were carefully cultivated. Aspergillus niger has high cellulose content, a characteristic that makes them play a key role in the fermentation process of cotton stalks [20].

2.2. Reagents

The reagents include the following: Potato Dextrose Agar (023,312), Potato Dextrose broth (014,250), Yeast Extract Peptone Dextrose (YPD) broth (5,193), De Man, Rogosa, and Sharpe (MRS) broth (02,841); nutrient broth (0,108) purchased from Haibo Biotechnology (Shandong, China); and enzyme-producing medium (1 g/100 mL CMCNa, 0.5 g/100 mL Peptone, 0.05 g/100 mL MgSO4, 0.1 g/100 mL K2HPO4). Other reagents were purchased from Luqiao (Beijing, China).

2.3. Fermentation Preparation

In total, 1 mL of Aspergillus niger (six strains) preservation solution was inoculated into 100 mL of sterile Potato Dextrose broth, and cultured at 170 rpm/min at 30 °C for 168 h. Simultaneously, the 0.1 mL of preservation solution was inoculated onto PDA for activation. Similarly, 1 mL of Candida utilize preservation solution was inoculated into a 100 mL sterile YPD broth, and incubated at 35 °C for 48 h. For Bacillus licheniformis, 1 mL of preservation solution was inoculated into a 100 mL sterile nutrient broth, and incubated at 35 °C for 48 h. The same procedure was followed for Lactobacillus casei, with 1 mL of preservation solution inoculated into a 100 mL sterile MRS broth, and incubated at 35 °C for 48 h.

2.4. Screening for Strains with High Cellulase Production

In the process of screening for high cellulase-producing strains of Aspergillus niger, an initial screening was conducted on six strains of Aspergillus niger. The six strains were inoculated onto Sodium Carboxymethyl Cellulose (CMC-Na) medium and incubated at 30 °C for 5 days. Subsequently, Congo red staining was performed for 10 min, followed by 2–3 washes with 1 mol/L NaCl solution. The diameter of the strains and the corresponding hydrolysis zone diameter were measured by Vernier Caliper (Chengguang, 0–200 mm, Nanjing, China). Then, the transparent zone to the colony diameter ratio of the six strains of Aspergillus niger was calculated to assess their cellulase degradation ability and growth rate. The filter paper enzyme activity (FPA) was assessed by measuring the weight loss rate of filter paper within a specified time period. The fungal strains were inoculated onto FPS medium and incubated at 30 °C, 80 rpm for 3, 5, and 7 days in a shaking incubator (Yiheng, HZQ-300C, Shanghai, China). After incubation, 1 mL of the culture was centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the filter paper was washed with a low-concentration mixture of hydrochloric and nitric acids to remove fungal cells. The paper was then centrifuged, washed with boiling water, dried at 105 °C, and weighed [21]. The strains selected from the initial screening were subjected to enzyme activity assays. Finally, the CMCase activity and FPA were measured using the DNS method to quantify the enzyme production capacity of each strain [22].

2.5. Experimental Design of Response Surface Methodology

Cotton stalks and corn flour are rich in carbon sources. By adjusting the cotton stalks and corn flour mass ratio (the additional amount of the auxiliary material is generally most suitable between 20% and 30%), nitrogen deficiency that limits microbial growth can be avoided, thereby providing an appropriate carbon-to-nitrogen ratio (C/N) to promote cellulose degradation and microbial community activity [23]. The inoculation amount directly influences the microbial density in the fermentation system. When the fungal inoculation amount is between 8 and 12%, a balance can be achieved between the cellulose degradation rate and fermentation cost [24]. Moisture content has a crucial effect on microbial metabolic activity, the mass transfer rate, and oxygen diffusion during fermentation. Moisture content should be maintained within the ideal range (60–80%) to significantly improve cellulose activity and lignin degradation rate in the straw [25].
Therefore, the mass ratio of cotton stalks to corn flour, inoculation amount, and moisture content was selected for a three-factor, three-level Box–Bhenken experiment to find the best conditions for cotton stalks’ solid-state fermentation., as shown in Table 1. The independent variables included the mass ratio of cotton stalks to corn flour, the inoculation amount, and the moisture content. The response variable was the crude fiber content in the cotton stalks, which was determined using a crude fiber analyzer (Ankom, ANKOM220, Macedon, NY, USA).

2.6. Determination of Cotton Stalk Composition During Solid-State Fermentation

The strain with the strongest enzyme activity obtained from the screening was inoculated into a solid fermentation medium and fermented at room temperature for 60 d. Every 10 d, the cotton stalk was removed for colony observation. The previously described method determined dry matter loss, and the lignin, cellulose, and hemicellulose contents were determined by the paradigm method [26,27].
The colonies were inoculated into 100 mL of enzyme-producing medium and incubated at 30 °C in a constant temperature shaker at 150 r/min for 1 h, followed by filtration. The fungal fluid was centrifuged at 4 °C, 8000 r/min for 10 min, and the supernatant was collected as the crude enzyme solution. Then, the activities of Laccase (Lac), carboxymethyl cellulase (CMCase), and Xylanase (Xylase) were determined by the ABTS method and the DNS method [28,29].

2.7. Mixed Microbial Solid-State Fermentation Experiment Design

The fermentation process in this study is divided into two stages. First, the Aspergillus niger strain HQXY was inoculated into cotton stalks for 30 days of aerobic fermentation, and the lignin, cellulose, and hemicellulose in cotton stalks were degraded initially [30]. After the completion of aerobic fermentation, the corresponding probiotic consortium was added at an inoculation ratio of 1:1:1 for anaerobic fermentation, further fiber degrading and intermediates of Aspergillus niger, and enhancing the conversion rate of nutrients in cotton stalks [31]. These processes are conducted at natural pH and temperature. As shown in Table 2, a total of 14 experimental groups were designed to investigate various combinations of microbial strains, including A + C (Aspergillus niger and Candida utilis), A + B + L (Aspergillus niger, Bacillus licheniformis, and Lactobacillus casei), and A + B + C + L (Aspergillus niger, Bacillus licheniformis, Candida utilis, and Lactobacillus casei), as well as inoculation ratios (5%, 10%, 15%) and anaerobic fermentation times (10, 20, 30 days).

2.8. Feed Quality Analysis

Following the stepwise fermentation of the cotton stalk bio-feed, a sensory assessment was conducted on-site according to German bio-feed sensory evaluation standards. The assessment criteria included smell (with a maximum of 14 points), texture (with a maximum of 4 points), and color (with a maximum of 2 points). The total score categorized the feed quality into four grades: excellent (16–20 points), good (10–15 points), fair (5–9 points), and spoiled (0–4 points). The PH between feed samples was determined by the PH meter (Leici, PHS-3C, China). Dry matter content was determined by Karga et al. with some modifications [32]. In total, 50 g was taken from each sample, dried in an oven at 65 °C for 48 h until a constant weight was achieved, then cooled and re-weighed. Crude protein was determined by the Kjeldahl method [33]. Gossypol was determined by High-performance liquid chromatography (HPLC) (Agilent, G1314-60186, Waldbronn, Germany).

2.9. Statistical Analysis

One-way ANOVA was analyzed using IBM SPSS Statistics 27 (IBM, Armonk, NY, USA) and Duncan’s multiple comparison tests to assess differences between groups. All parameters were replicated three times. Results are expressed as means and standard deviations. p < 0.05 was considered statistically significant. Stacked bar charts were created using GraphPad Prism 9.5 (GraphPad, San Diego, CA, USA). Point line graphs, contour plots, and response surface plots were generated using Origin Pro (OriginLab, Northampton, MA, USA). Response surface optimization design was performed using Design-Expert 13.0 (Stat-Ease Inc., Minneapolis, MN, USA).

3. Results

3.1. Determination of High Cellulase-Producing Strains

As shown in Table 3, an initial screening of six strains of Aspergillus niger was conducted using Congo red staining, and it can be observed that HQXY exhibited the largest colony diameter, suggesting that it has the strongest cellulase production capacity. To further confirm the screening results, a filter paper enzyme activity re-screening test was performed on the six strains of Aspergillus niger. As shown in Table 4, from the third day of cultivation, HQXY consistently exhibited the highest weight loss rate on the filter paper, reaching its maximum on day seven, significantly higher than the other five strains. Based on the enzyme activity measurement results (Table 5), it can be observed that the cellulase activity and filter paper enzyme activity of the six strains of Aspergillus niger show significant differences (p < 0.05), and HQXY’s cellulase activity and filter paper enzyme activity are higher than those of the other five strains. Therefore, it can be concluded that HQXY exhibited the highest cellulase activity and was selected as the final degrading fungus to participate in the solid-state fermentation process [34]. By selecting high cellulase-producing strains of Aspergillus niger, instability during the feed fermentation process was avoided, and costs and resources were saved, thereby laying the foundation for improving the nutritional value of cotton stalk feed [35].

3.2. Verification Results of the Box–Bhenken Experiment Design

Based on the regression analysis of the crude fiber response surface coefficients, the regression equation of the model was as follows: Y = 304.86760 + 0.54788A + 2.08838B + 3.36100C − 6.61250AB + 3.75075AC − 6.95775BC − 11.82655A2 − 4.14905B2 − 5.47080C2. As shown in Table S1, the model p < 0.01 was significant. At the same time, the lack of fit p = 0.5975 was not significant, suggesting that the regression equation has a good fit. According to the analysis of the residual normal plot (Figure S1A), the data points are distributed along a theoretical straight line without significant skewness or kurtosis, thereby satisfying the residual normality hypothesis. Secondly, all points in the residual-predicted plot (Figure S1B) are randomly distributed above and below the zero-value line, and no evidence of heteroscedasticity is observed, indicating that the hypothesis of homogeneity of variance for the model is valid. Furthermore, the multiple correlation coefficient R2 = 0.9752, and the adjusted R2 = 0.9433, close to 1, indicate a good fit with the actual data (Table S2).
The effects of factor interactions on the response values were analyzed using 3D response surface plots. As shown in Figure 1, response surface analysis was conducted to examine the effects of the cotton stalk-to-corn flour ratio, inoculation amount, and moisture content on the crude fiber content of cotton stalks. The contour lines appeared elliptical, indicating a significant interaction between the cotton stalk-to-corn flour ratio and inoculation amount (Figure 1A). As the crude fiber content decreased, the cotton stalk-to-corn flour ratio and inoculation amount increased, with a steeper slope on the surface, suggesting that these two factors significantly impact the crude fiber content (Figure 1B). The cotton stalk-to-corn flour ratio contour lines and moisture content were also elliptical, demonstrating a significant interaction between these two factors (Figure 1C). When both the cotton stalk-to-corn flour ratio and moisture content were reduced, the crude fiber content showed a decreasing trend (Figure 1D). The crude fiber content decreased as the inoculation amount decreased, and it initially increased and then decreased with the increase in moisture content (Figure 1F), with a significant interaction between these two factors (Figure 1E). The optimal conditions determined through binomial analysis were A, B, and C at the −1.00 level, corresponding to a cotton stalk-to-corn flour mass ratio of 5:1, an inoculum amount of 8%, and a moisture content of 65%. Under these conditions, the predicted crude fiber content was 267 g·kg−1. After 30 days of fermentation, the verification experiment showed a crude fiber content of 271 g·kg−1, representing a 34% reduction from the initial value of 410.61 g·kg−1 under the solid-state fermentation conditions, confirming that the experimental design was reasonable and accurate.

3.3. Degradation of the Cotton Stalk by Aspergillus niger

Reducing cellulose, hemicellulose, and lignin levels in feed is crucial for enhancing animal digestibility and growth performance. Investigating the degradation of lignin, cellulose, and hemicellulose in cotton stalks by Aspergillus niger can provide deeper insights into its ability to break down fibrous components. Compared with cotton stalks that were not inoculated with Aspergillus niger (Figure 2A), those inoculated (Figure 2B) exhibited significant degradation of cellulose and hemicellulose, while lignin degradation was relatively slower. After 60 days of fermentation, cellulose, hemicellulose, and lignin contents reached their lowest levels, at 28.5%, 7.9%, and 11.58%, respectively. The dry matter loss also increased to 20.72%. These results indicate that Aspergillus niger effectively degrades cellulose and hemicellulose, converting these components into microbial metabolic products or releasing them as carbon dioxide, thereby reducing dry matter content [36]. In contrast, lignin degradation was less pronounced due to its complex and unique structural characteristics [37]. Therefore, the biocatalytic degradation of lignin can be enhanced through the combination of lignin-degrading enzymes with auxiliary enzymes or via the optimization of fermentation conditions. For instance, lignin can be efficiently degraded through the addition of polysaccharide monooxygenases and lignin-degrading enzymes, coupled with pretreatment methods such as acid-base treatment or oxidation [38,39].
Figure 2C, Figure 2D and Figure 2E present the activity profiles of Lac, Xylase, and CMCase produced by Aspergillus niger during fermentation, respectively. Lac activity increased continuously throughout the fermentation period, reaching a maximum of 72.37 U·mL−1 by 60 days post-inoculation. This trend is closely related to the dynamic requirements of lignin degradation. Lac disrupts the cross-linking structure of lignin by oxidizing phenolic compounds, and its sustained high activity indicates that lignin has not been completely degraded in the later stages of fermentation, which may become a limiting factor for cellulose exposure [40]. Xylase activity exhibited a rapid increase during the initial 20 days of fermentation, peaking at 2786.39 U·mL−1 on day 20, after which a gradual decline was observed. Nevertheless, the activity remained relatively high (1215.69 U·mL−1) even at 60 days post-inoculation. This suggests that hemicellulose degradation primarily occurs during the mid-fermentation period, while residual xylan may continue to inhibit the interaction between cellulase and the substrate through steric hindrance [41]. CMCase activities demonstrated a sharp increase during the early fermentation phase (first 30 days), attaining peak levels of 291.16 U·mL−1, by day 30. Subsequently, a pronounced decline in activity was observed, with the lowest values recorded by day 60. This trend is directly related to substrate consumption. Initially, cellulase efficiently hydrolyzed crystalline cellulose through synergistic actions (such as β-glucosidase and endo-β-glucanase), but as accessible cellulose decreased and enzyme proteins underwent thermal inactivation, CMCase activity significantly decreased, leading to a reduction in cellulose conversion efficiency [42].

3.4. The Results of Sensory Evaluation

For every sample group, sensory evaluations centered on color, texture, and odor were conducted. Figure 3 shows that all samples had scores between 10 and 19, with the (A + B + L) microbial blend receiving the highest sensory scores and being classified as high-quality feed. This better performance is probably caused by Aspergillus niger’s early aerobic fermentation of cotton stalks, which partially breaks down lignin, cellulose, and hemicellulose and turns them into carbohydrates [43]. Following this, the introduction of Lactobacillus casei and Bacillus licheniformis initiates anaerobic fermentation. Lactobacillus casei metabolizes the available carbohydrates to produce lactic acid, which significantly lowers the pH of the cotton stalk feed [44]. This acidification creates a favorable environment that extends the shelf life and enhances the feed’s palatability through the development of a sour, pleasant aroma. Additionally, Bacillus licheniformis plays a dual role by suppressing the growth of spoilage bacteria and by fostering the proliferation of Lactobacillus casei [45]. In contrast, the relatively low microbial consortia scores of A + B + C + L and A + C can be attributed to the fact that ethanol produced by Candida utilis disrupts the metabolic balance through the inhibition of extracellular enzyme activity in Aspergillus niger and bacteria, thereby reducing the decomposition efficiency of lignocellulose. Simultaneously, the carbon source competition between Candida utilis, Aspergillus niger, and functional strains (B and L) can weaken the metabolic activity of cellulose-degrading strains, which results in a reduced enzyme production efficiency of the fermentation system [46].

3.5. The Results of Nutritional Value

The pH values, crude protein content, dry matter content, and peanut content of the 13 experimental samples were measured (Table 6). By measuring these physicochemical indicators, the significant effects of stepwise fermentation with a composite microbial community can be verified and the fermentation effect of mixed microbial fermentation on cotton stalk feed can be assessed. The pH of ruminant animal feed should be maintained between 4.0 and 5.0, as this range is more beneficial for digestion and absorption while inhibiting the growth of certain pathogens in the animal’s digestive tract [47]. The results of the pH measurements demonstrate that, except for experimental group 4, all other experimental groups met the feed standards. The measurement results for dry matter content and crude protein content demonstrate that the control group (group 14) had the lowest dry matter content, while the experimental group 13 had the highest. Furthermore, the crude protein content in experimental groups 3, 4, 7, and 9 was lower than that of the group 14, while the crude protein content in all other experimental groups was higher than that of the group 14. Notably, the crude protein content in experimental group 13 was significantly higher than that of the group 14, increasing by 13.93%. Gossypol is an antinutritional factor in cotton, which, although possessing certain antibacterial and antioxidant properties, has a toxic effect on ruminant animals and may negatively affect animal health, leading to loss of appetite, indigestion, or toxic reactions [48]. The results of gossypol measurements demonstrate that, compared with the control group, the gossypol content in all 14 experimental samples was reduced (with an initial gossypol content of 137 mg·kg−1), with experimental group 13 showing the lowest gossypol content of 15.50 mg·kg−1, demonstrating the best degradation effect. Through a comprehensive analysis of the 13 feed groups, the feed product from group 13 exhibited the best quality, improving the nutritional value of cotton stalk feed and reducing the gossypol content, ensuring its safety.

4. Discussion

4.1. Primary and Re-Screening of Aspergillus niger

The Aspergillus niger strains isolated from the Xinjiang cotton soil were prioritized for dual-parameter screening (e.g., cellulase activity × growth rate) to overcome the limitations of single-method approaches. Although the Congo red staining method is widely used for cellulase detection, its accuracy is affected by starch interference and pigment degradation [49]. To address this issue, a complementary filter paper strip test was introduced, simulating natural cellulose matrices and quantifying extracellular enzyme efficacy [50]. Furthermore, the quantification of CMCase and FPase may enhance the accuracy of strain selection. Through the integration of the above methods, high cellulase-producing strains can be identified in a more systematic and comprehensive manner.

4.2. Degradation of Cruder Fiber Content Using Response Surface Methodology

The fiber degradation rate reached 34% through response surface optimization, which exceeded the 24.38% reported by Ti et al. [51], potentially due to the synergistic effect of corn flour (starch water absorption and gelatinization softening fiber) and the promotion of mycelial penetration under a 65% water condition [52]. However, the observed dry matter loss of 20.72% remained higher than that in anaerobic fermentation systems, and further optimization of carbon fixation pathways (such as the addition of azotobacter or biochar adsorption) is required to improve biomass utilization. The substrate ratio (cotton stalk: corn meal = 5:1) resulted in the activities of Xylase and CMCase peaking at 2786.39 U·mL−1 and 291.16 U·mL−1 at 20 and 30 days, respectively. However, a sudden drop in CMCase activity (<100 U·mL−1 at 60 days) revealed resistance in the cellulose crystalline region. This contrasts with the strategy employed by Thermobifida fusca and other thermophilic bacteria to achieve continuous degradation through heat-stabilized cellulase, suggesting that Aspergillus niger must be combined with physical pretreatment (such as steam blasting) to enhance substrate accessibility [53]. The degradation rate of cellulose and hemicellulose was significantly higher than that of lignin, which directly relates to the complex β-O-4 bond and aromatic ring structure of lignin. Although Lac activity continued to rise to 72.37 U·mL−1, its low catalytic efficiency on non-phenolic lignin structures resulted in lignin residue, under the limitations of most filamentous fungi dependent on a single Lac system [54]. In contrast, white rot bacteria (e.g., Phanerochaete chrysosporium) secrete lignin peroxidase (LiP), which can target the cleavage of the β-O-4 bond and increase the lignin degradation rate to 30%. This indicates that Aspergillus niger needs to combine exogenous LiP or photocatalytic oxidation to overcome these limitations [55].

4.3. Fungal and Bacterial Stage Fermentation System

This study significantly improved the quality and nutritional value of cotton stalk feed through a phased fermentation strategy involving a complex microbial community (A + B + L). Aspergillus niger initiated the degradation of cotton stalk lignocellulose by secreting cellulase and hemicellulase, with the carbohydrates produced serving as a substrate for subsequent anaerobic fermentation. This process aligns with the mechanism employed by Fadel et al. [56] in fermenting animal feed with Aspergillus fumigatus. Subsequently, Lactobacillus casei produces lactic acid by metabolizing carbohydrates, lowering the pH to the optimal range of 4.0–5.0. This not only inhibits the proliferation of putrid bacteria but also enhances the palatability of the feed through acidification. Additionally, Bacillus licheniformis inhibits spoilage bacteria by secreting antimicrobial peptides, while promoting the colonization of Lactobacillus casei, thus resulting in a positive interaction cycle [57]. In contrast, the bacterial assemblage containing Candida prion-producing bacteria (A + C and A + B + C + L) performed poorly, and the crude protein content was not significantly improved. This result contradicts the yeast–Bacillus synergistic protein production phenomenon reported by Sheng et al. [58], which may be attributed to the rapid consumption of soluble sugars by yeast during the early aerobic stage, leading to intensified competition for carbon sources in the subsequent anaerobic stage. Regarding gossypol degradation, the degradation rate in experimental group 13 was 88.69%, which was superior to the rate reported by Zhang et al. ([59], 81.83%) using single bacteria solid-state fermentation. Lac, potentially derived from Aspergillus niger, formed a cascade catalytic system with peroxidase from Bacillus, thereby increasing the degradation rate. Although the complex bacterial community can improve the nutritional index, the effect on crude protein content is not significant. Therefore, in the future, genetic engineering or protein recombination technologies could be employed to enhance bacterial community activity and protease secretion, thereby improving the protein content of the feed [60].

4.4. Prospects

During fermentation, the reduction in pH plays a crucial role in inhibiting the growth of harmful microorganisms and prolonging preservation time. However, various factors can influence storage stability, which may trigger secondary fermentation or alter microbial activity, ultimately impacting the quality and stability of silage [61]. Therefore, long-term conservation must be achieved through microbial regulation, environmental control, and monitoring technologies. Although the degradation efficiency has improved, long-term enzyme stability (>60 days) remains unresolved, which may limit industrial scalability. Future work should explore immobilization technology to extend enzyme activity. Immobilization technology can significantly enhance the stability and reusability of enzymes through their immobilization on a carrier. For instance, developing new nanomaterial carriers or microencapsulation technologies can further improve the resistance and reuse of enzymes [62]. Simultaneously, the multi-enzyme co-immobilization system has been explored to simplify the synthesis steps of complex products [63]. Additionally, transcriptomic and metabolomic analyses targeting microbial communities should also be involved. Metabolomics analysis, combined with high-throughput sequencing, can dynamically track gene expression patterns across different species and uncover the synergistic regulatory mechanisms of key metabolic pathways such as cellulose degradation and amino acid synthesis [64]. This multi-omics integration strategy provides data-driven insights for the optimization of microbial communities. Simultaneously, genomics and metabolomics analyses can help identify unknown by-products produced during feed fermentation and assess their potential safety risks, thereby further improving the nutritional value and safety of the feed [65].

5. Conclusions

The results of this study offer significant technical support for cotton stalk feed and demonstrate the potential for application in large-scale feed production. Future research could further enhance feed quality by optimizing fermentation time or investigating additional microbial populations. Additionally, the scalability of large-scale feed production and its integration with existing livestock feeding systems remain significant concerns. The exploration of alternative probiotic combinations or the optimization of fermentation processes for various agricultural wastes could serve as key avenues for future research. Through these efforts, safer and more nutritious feed products are expected to be developed, promoting the efficient use of agricultural waste and the sustainable development of livestock.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation11030124/s1. Table S1: Variance analysis of the crude fiber content response surface results; Table S2: Box–Behnken response surface design and results. Figure S1: Residual analysis and normal verification of the crude fiber content response surface results.

Author Contributions

Conceptualization, Y.L., M.H. and K.L.; methodology, K.L. and K.G.; software, K.L. and Y.X.; formal analysis, Y.X.; investigation, K.L., K.G. and Y.X.; resources, W.C.; data curation, M.H. and K.L.; writing—original draft preparation, K.L. and Y.X.; writing—review and editing, Y.L., M.H. and K.L.; visualization, K.L.; supervision, Y.L., M.H. and Y.X.; project administration, Y.L. and M.H.; funding acquisition, W.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Task Special Project of Xinjiang Uygur Autonomous Region, grant number 2022B02042; Supported by the sub tasks of the Key Research and Development Program of Xinjiang Uygur Autonomous Region, grant number 2022B02056-2; Supported by the sub tasks of Xinjiang Academy of Agricultural Sciences Agricultural Science and Technology Innovation Stabilization Support Program, grant number xjnkywdzc-2023005-2.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Acknowledgments

Materials used for experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Fermentation 11 00124 g001
Figure 1. Influence of different factors on crude fiber content. Influence of the cotton stalk to corn flour mass ratio and inoculation amount on crude fiber content. (A) Contour graph. (B) 3D surface. Influence of the cotton stalk to corn flour mass ratio and moisture content on crude fiber content. (C) Contour graph. (D) 3D surface. Influence of the inoculation amount and moisture content on crude fiber content. (E) Contour graph. (F) 3D surface.
Figure 1. Influence of different factors on crude fiber content. Influence of the cotton stalk to corn flour mass ratio and inoculation amount on crude fiber content. (A) Contour graph. (B) 3D surface. Influence of the cotton stalk to corn flour mass ratio and moisture content on crude fiber content. (C) Contour graph. (D) 3D surface. Influence of the inoculation amount and moisture content on crude fiber content. (E) Contour graph. (F) 3D surface.
Fermentation 11 00124 g001
Fermentation 11 00124 g002
Figure 2. Degradation of cotton stalk components and enzyme activity profiles by Aspergillus niger. (A) Cotton stalk without inoculation with Aspergillus niger. (B) Cotton stalk inoculated with Aspergillus niger. (C) Lac activity profiles. (D) Xylase activity profiles. (E) CMCase activity profiles.
Figure 2. Degradation of cotton stalk components and enzyme activity profiles by Aspergillus niger. (A) Cotton stalk without inoculation with Aspergillus niger. (B) Cotton stalk inoculated with Aspergillus niger. (C) Lac activity profiles. (D) Xylase activity profiles. (E) CMCase activity profiles.
Fermentation 11 00124 g002
Fermentation 11 00124 g003
Figure 3. Results of the sensory evaluation of cotton stalks under different Treatments. Excellent (16–20 points), good (10–15 points), fair (5–9 points), and spoiled (0–4 points).
Figure 3. Results of the sensory evaluation of cotton stalks under different Treatments. Excellent (16–20 points), good (10–15 points), fair (5–9 points), and spoiled (0–4 points).
Fermentation 11 00124 g003
Table 1. Factors and levels of Box–Behnken design.
Table 1. Factors and levels of Box–Behnken design.
FactorsLevel
−110
A: Cotton stalk to corn flour mass ratio5:14:13:1
B: Inoculation amount8%10%12%
C: Moisture content65%70%75%
Table 2. Mixed microbial fermentation experimental design.
Table 2. Mixed microbial fermentation experimental design.
GroupMicrobial Strain CombinationInoculation Amount (%)Fermentation Time (d)
1A + B + L1020
2A + B + C + L1010
3A + B + L510
4A + C520
5A + B + C + L1520
6A + B + L530
7A + C1010
8A + B + L1530
9A + C1520
10A + B + C + L520
11A + B + C + L1030
12A + B + L1510
13A + B + L1030
14A530
A: Aspergillus niger; B: Bacillus licheniformis; C: Candida utilis; L: Lactobacillus casei.
Table 3. Transparent circle and cell diameter of Six Aspergillus niger strains.
Table 3. Transparent circle and cell diameter of Six Aspergillus niger strains.
StrainD (mm)d (mm)D/dA
L32.59 ± 0.402.40 ± 0.371.080.518
L48.14 ± 0.763.42 ± 0.852.381.628
HQZD12.50 ± 3.124.38 ± 0.982.852.500
HQXY16.23 ± 2.393.46 ± 0.684.693.246
Z310.36 ± 1.664.24 ± 0.822.442.072
Z49.24 ± 2.505.22 ± 1.021.771.848
The relative specific activity (A) of the enzyme = diameter of the clear zone/number of days (5 days).
Table 4. The weight loss rate of filter paper strips for six Aspergillus niger strains.
Table 4. The weight loss rate of filter paper strips for six Aspergillus niger strains.
Strain Weight Loss Rate
3 d5 d7 d
L310.72% ± 0.10 a12.56% ± 0.43 a15.67% ± 1.64 a
L49.94% ± 0.24 a34.85% ± 0.78 c60.68% ± 1.14 c
HQZD25.27% ± 0.92 b46.28% ± 1.17 d72.36% ± 1.51 d
HQXY26.35% ± 0.94 b51.82% ± 0.81 d82.49% ± 1.87 e
Z311.56% ± 0.73 a23.34% ± 1.06 b40.34% ± 1.18 b
Z48.45% ± 1.20 a29.56% ± 0.85 b44.78% ± 2.12 b
Weight loss rate (%) = initial weight -dried weight /initial weight. Different lowercase letters represent significant differences at p < 0.05 based on the analysis of variance in different strains of the same cultural time.
Table 5. The enzyme activity for six Aspergillus niger strains.
Table 5. The enzyme activity for six Aspergillus niger strains.
StrainsCMCase (U.mL−1)FPase (U.mL−1)
L3150.66 ± 8.19 a80.30 ± 4.71 a
L4203.29 ± 6.40 b114.29 ± 8.50 b
HQZD243.74 ± 21.78 c133.29 ± 11.26 c
HQYX255.35 ± 9.94 c151.69 ± 8.24 d
Z3144.83 ± 31.51 a70.39 ± 8.52 a
Z4126.99 ± 8.71 a50.23 ± 6.95 a
Different lowercase letters represent significant differences at p < 0.05 based on the analysis of variance in different strains of the same cultural time.
Table 6. Results of the nutritional index of cotton stalks under different treatments.
Table 6. Results of the nutritional index of cotton stalks under different treatments.
GrouppHCrude Protein Content (%)Dry Matter Content (%)Gossypol Content (mg·kg−1)
14.21 ± 0.01024.9 ± 0.07326.89 ± 0.04023.19 ± 0.015
24.89 ± 0.05823.1 ± 0.06526.08 ± 0.03548.18 ± 0.030
34.52 ± 0.05822.8 ± 0.04726.31 ± 0.03225.31 ± 0.035
45.85 ± 0.05822.1 ± 0.03025.84 ± 0.05566.18 ± 0.361
54.77 ± 0.02123.5 ± 0.03326.08 ± 0.03047.46 ± 0.076
63.92 ± 0.01525.3 ± 0.05526.83 ± 0.07022.65 ± 0.153
74.89 ± 0.02122.6 ± 0.02125.69 ± 0.02561.69 ± 0.038
83.97 ± 0.01225.5 ± 0.04626.74 ± 0.02017.68 ± 0.026
94.94 ± 0.0122.3 ± 0.05325.99 ± 0.02564.13 ± 0.076
104.82 ± 0.01022.9 ± 0.03926.21 ± 0.04048.19 ± 0.030
114.88 ± 0.01023.6 ± 0.04426.17 ± 0.02544.50 ± 0.057
124.12 ± 0.01025.7 ± 0.03026.67 ± 0.03025.93 ± 0.021
134.00 ± 0.01526.0 ± 0.01127.01 ± 0.02015.50 ± 0.015
146.81 ± 0.02022.82 ± 0.01725.55 ± 0.07282.30 ± 0.055
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Li, K.; Xu, Y.; Guo, K.; Cui, W.; Li, Y.; Hou, M. Improving the Nutritional Value and Safety of Cotton Stalk Feed via Response Surface Methodology and Co-Fermentation Techniques. Fermentation 2025, 11, 124. https://doi.org/10.3390/fermentation11030124

AMA Style

Li K, Xu Y, Guo K, Cui W, Li Y, Hou M. Improving the Nutritional Value and Safety of Cotton Stalk Feed via Response Surface Methodology and Co-Fermentation Techniques. Fermentation. 2025; 11(3):124. https://doi.org/10.3390/fermentation11030124

Chicago/Turabian Style

Li, Kunyi, Yuansheng Xu, Kai Guo, Weidong Cui, Yang Li, and Min Hou. 2025. "Improving the Nutritional Value and Safety of Cotton Stalk Feed via Response Surface Methodology and Co-Fermentation Techniques" Fermentation 11, no. 3: 124. https://doi.org/10.3390/fermentation11030124

APA Style

Li, K., Xu, Y., Guo, K., Cui, W., Li, Y., & Hou, M. (2025). Improving the Nutritional Value and Safety of Cotton Stalk Feed via Response Surface Methodology and Co-Fermentation Techniques. Fermentation, 11(3), 124. https://doi.org/10.3390/fermentation11030124

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