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Article

Construction of Microbial Consortium to Enhance Cellulose Degradation in Corn Straw during Composting

by
Jie Li
1,2,
Juan Li
2,3,
Ruopeng Yang
4,
Ping Yang
2,
Hongbo Fu
2,
Yongchao Yang
2 and
Chaowei Liu
1,*
1
College of Life Science, China West Normal University, Nanchong 637000, China
2
College of Biological and Agricultural Sciences, Honghe University, Mengzi 661100, China
3
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
4
College of Chemistry and Resources Engineering, Honghe University, Mengzi 661100, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 2107; https://doi.org/10.3390/agronomy14092107
Submission received: 16 August 2024 / Revised: 12 September 2024 / Accepted: 13 September 2024 / Published: 16 September 2024
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
Browse Figures
Versions Notes

Abstract

:
The improper treatment of crop straw not only leads to resource wastage but also adversely impacts the ecological environment. However, the application of microorganisms can accelerate the decomposition of crop straw and improve its utilization. In this study, cellulose-degrading microbial strains were isolated from naturally decayed corn straw and screened using Congo red staining, along with assessing variations in carboxymethyl cellulase (CMCase) activity, filter paper enzyme (FPase) activity and β-glucosidase (β-Gase) activity, as well as the degradation rate. The eight strains, namely Neurospora intermedia isolate 29 (A1), Streptomyces isolate FFJC33 (A2), Gibberella moniliformis isolate FKCB-009 (A3), Fusarium fujikuroi isolate EFS3(2) (A4), Fusarium Fujikuroi isolate FZ04 (A5), Lysine bacillus macroides strain LNHL43 (B1), Bacillus subtilis strain MPF30 (B2) and Paenibacilli lautus strain ALEB-P1 (C), were identified and selected for microbial strain consortium design based on their high activities of CMCase, FPase and β-Gase. The fungi, bacteria and actinomycete strains were combined without antagonistic effects on corn straw decomposition. The results showed the A2B2 combination had a significantly higher FPase at 55.44 U/mL and β-Gase at 25.73 U/mL than the other two strain combinations (p < 0.05). Additionally, the degradation rate of this combination was 40.33%, which was considerably higher than that of the other strains/consortia. The strain combination A4B2C also had superior enzyme activity, including CMCase with a value of 35.03 U/mL, FPase with a value of 63.59 U/mL and β-Gase with a value of 26.15 U/mL, which were significantly different to those of the other three strain combinations (p < 0.05). Furthermore, seven single microbial strains with high cellulase activities were selected to construct various microbial consortiums for in situ composting in order to evaluate their potential. Taken as a whole, the results of composting, including temperature, moisture content, pH, E4/E6 value and seed germination index, indicated that the microbial strain consortium consisting of Neurospora intermediate isolate 29, Fusarium fujikuroi isolate EFS3(2), Fusarium fujikuroi isolate FZ04, Lysinibacillus macrolides, Lysinibacillus sphaericus, Bacillus subtilis and Paenibacillus lautus was advantageous for corn straw decomposition and yielded high-quality compost. The screened flora was able to effectively degrade corn straw. This study provides a novel solution for the construction of a microbial consortium for the composting of corn straw.

1. Introduction

Crop straw is a valuable biological resource with multifunctionality and renewability, but its actual utilization rate is still relatively low [1]. In 2022, global crop straw production reached 3 billion tons, and more than 60% of this crop straw, corn straw in particular, was randomly discarded and burnt, which represents a serious environmental problem, as the utilization rate of crop straw is thus less than 40% [2,3]. In China, the comprehensive utilization rate of straw is relatively low, with current methods being relatively simplistic and not very advanced, and few high-value utilization techniques are available. Technology and equipment limitations in straw processing contribute to the annual disposal or incineration of large amounts of straw [4]. Burning straw not only leads to a dramatic increase in surface temperature, which can kill soil microorganisms and reduce soil moisture loss, affecting soil microbial and enzyme activity, but also contributes to higher carbon dioxide concentrations, exacerbating the greenhouse effect and causing serious environmental problems. Addressing the environmental pollution issues and finding a solution to inefficient corn straw degradation have become quite important [5].
Straw is rich in cellulose and hemicellulose and contains abundant organic matter and nutrients, such as N, P, K, Mg and Ca, which are essential for crop growth. Returning straw to fields as fertilizer can reduce reliance on chemical fertilizers, enhance the content of organic matter in the soil and promote an increase in crop yield. Composting is also a promising organic waste management method [1]. It is possible to convert organic waste into fertilizer and reduce pathogens [6]. Mature compost can be used for plant growth and soil improvement [7]. Composting is a biochemical process mediated by a variety of microorganisms that destroy pathogens and recycle nutrients. It was found that returning straw compost to fields provides carbon and energy sources for soil microorganisms and plants, maintains soil microbial community diversity and improves soil physicochemical properties, thereby promoting greater nutrient uptake by cowpea root systems and increasing the chlorophyll content and antioxidant capacity of plant leaves, thereby supporting the growth and development of crops such as cowpea [8]. However, the uncontrolled accumulation of straw can lead to the production of leachate, slow degradation rates, large emissions of greenhouse gases and the waste of land resources, all of which have serious impacts on the environment and sustainable economic development. Corn straw is mainly composed of cellulose, hemicellulose and lignin, which decompose and degrade on their own with difficulty [9]. Currently, physical, chemical and biological treatments can be used for corn straw degradation [10]. Biological treatment includes microbial fermentation and enzymatic hydrolysis, which are environmentally friendly and economical alternatives to other methods [11]. The use of enzymes or microorganisms for the biological pretreatment of straw has significant advantages, such as reduced environmental impact, low energy requirements, efficient lignin degradation and the minimized production of inhibitory compounds, such as furfural, hydroxymethylfurfural and organic acids, during enzymatic hydrolysis and fermentation [12].
At present, utilizing microorganisms that secrete cellulase to degrade corn straw is an effective method. Microorganisms used to degrade lignocellulose, such as bacteria, fungi and actinomycetes, have received increasing attention [13]. Efficient cellulose-degrading strains of fungi, bacteria and actinomycetes can be screened through appropriate isolation methods [14]. Although microorganisms can effectively degrade lignin, they also consume cellulose and hemicellulose for their growth, resulting in the significant loss of fermentable sugars. There are still many challenges in industrial applications [15]. Although the degradation characteristics of enzyme production by microorganisms and the biodegradation process can be utilized to achieve resource utilization by fermenting crop straw under optimal conditions, due to differences in environmental requirements, low enzyme activity and long action time, there are relatively few microorganisms capable of degrading lignin in practical processes, limiting the further expansion of microbial treatment [16]. The variability of external conditions, the complexity of cellulose structure and the challenges associated with hydrolytic acidification contribute to differing degradation capabilities among microorganisms. Furthermore, even the same microorganism may exhibit heterogeneous degradation abilities under varying conditions. Therefore, how to select suitable and efficient microbial degradation bacteria and efficiently utilize them has become a problem in research.
Previous research has unraveled that the microorganisms involved in the fermentation of corn straw mainly originate from soil and decayed crop straw [17,18]. Song et al. [3] found that the fungus Coprinus comatus could effectively degrade the lignocellulose of corn straw, achieving a degradation rate of 38.66%. Li et al. [13] isolated two strains from soil and found that T. afroharzianum showed remarkable cellulose degradation efficiency for corn straw compared to T. longibrachiatum. At present, most studies focused on corn straw degradation by single strain, which had some limitations such as high environmental sensitivity [3,19,20]. Yu et al. [21] reported that microbial consortia had a higher decomposition efficiency and cellulase activity compared to a single strain. The varied cellulase production abilities among different microbial strains suggest that combining strains with higher cellulase production could enhance corn straw degradation [22]. Sharker et al. [23] reported that pretreating rice straw with a microbial consortium increased the amorphous cellulose ratio compared to a single strain. Pan et al. [24] found that screened microbial consortia efficiently degraded rice straw at 35 °C. When straw is returned to fields, the addition of the exogenous degradation bacterium ZJW-6, along with a composite bacterial system comprising both exogenous and endogenous degradation bacteria, improved the degradation rate of rice straw [25]. Liu et al. [26] constructed a composite bacterial system using three cellulose-degrading actinomycetes and found that this system significantly outperformed single strains in degrading corn straw. Phukongchai et al. [27] added exogenous straw microorganisms to the returned straw which accelerated the decomposition of straw in the soil and significantly increased the activity of soil microorganisms. Overall, the enzyme activity of the composite bacterial system constructed from multiple single strains was significantly higher than that of a single strain. The synergistic interactions among various strains enable more efficient straw degradation, making the development of microbial strain consortia for corn straw degradation a central focus of current research.
Therefore, it is urgent to find the most efficient microbial consortia for corn straw decomposition. In this study, we screened cellulose-degrading microbial strains based primarily on the size of the transparent circle using the Congo red staining method. Carboxymethyl cellulase (CMCase) catalyzes the breakdown of cellulose chains into smaller fragments, facilitating the saccharification of cellulose. During the degradation of corn straw, CMCase activity directly affects both the rate and extent of cellulose decomposition, making it a critical indicator of cellulose degradation efficiency. β-Glucosidase is mainly involved in the breakdown of oligosaccharides, such as cellobiose, into glucose, which is the final step in the cellulose degradation process and a key step in saccharification. The activity of filter paper enzymes serves as an indicator of overall activity of cellulases because they mimic the structure of natural cellulose. By measuring the activity of filter paper enzymes, one can assess the ability of cellulases to degrade complex fiber structures [10,28,29].
Furthermore, the microbial strains were rescreened based on carboxymethyl cellulase activity, filter paper enzyme activity and β-glucosidase activity. Simultaneously, strain identification was performed through morphological observation, 16S rRNA and ITS sequencing homology. The screened high-efficiency cellulose-degrading microbial strains were then tested for compatibility to ensure the absence of antagonistic interactions. Finally, an in situ composting experiment was conducted to evaluate the degradation efficiency of different microbial strain combinations. This approach aims to provide a theoretical basis and practical reference for addressing environmental pollution and developing efficient microbial degradation methods for corn straw resources.

2. Materials and Methods

2.1. Isolation and Purification Microbial Strains

Decayed corn straw was collected from five locations in a field of Caoba Village, Mengzi City, Yunnan Province, China. After air drying, the corn straw was cut into pieces (≤1 cm) and stored in sterilized polyethylene bags for subsequent screening. The dried corn straw was then mixed thoroughly with potato dextrose agar (PDA) medium and incubated at 28 °C for 3–5 days. Single colonies were isolated through multiple rounds of cultivation and purification. These were subsequently inoculated into improved PDA culture medium, beef extract peptone culture medium and GAUZE’s Medium No. 1. Bacteria, fungi and actinomycete strains were isolated based on standard separation and purification procedures.

2.2. Screening and Identification of Microbial Strains

2.2.1. Primary Screening

Following the method of Das et al. [30] with minor modifications, single colonies were inoculated onto beef extract peptone medium and GAUZE’s Medium No. 1 and incubated at 28 °C for 3–5 days. The microbial colonies were then transferred to Congo red medium and incubated at 28 °C for another 3–5 days. The colonies were immersed in a 1 mg/mL Congo red staining solution for 30 min. After staining, the Congo red was removed and the colonies were decolorized with 1 mol/L NaCl for 20 min. The diameter ratio of the transparent circle to the corresponding colony (D/d) was then measured [31].

2.2.2. Rescreening

Based on the method of Lu et al. [32] with minor modifications, a microbial suspension of the single strains obtained from primary screening was prepared and inoculated into a liquid enzyme production medium. The cultures were grown in a shaker at 120 rpm and 30 °C for 72 h. The culture solution was then centrifuged at 4000 rpm for 10 min and the supernatant was used as the crude enzyme solution to determine carboxymethyl cellulase activity, filter paper enzyme activity and β-glucosidase activity.
Following the method of Li et al. [14] with minor modifications, the microbial strains were inoculated onto a culture medium containing corn straw at 28 °C for 30 days, with a control medium containing 5% sterile water. The degradation rate was determined every 10 days. Based on enzyme activity and degradation rate, five fungi strains, two bacteria strains and one actinomycete strain were ultimately selected from the isolated microbial strains.

2.3. Sequencing and Identification

The DNAs of five fungi, two bacteria and one actinomycete were extracted by a DNA extraction kit (DP712, TIANGEN, Beijing, China) following the manufacturer’s instructions. Sequencing and identification were conducted by Beijing Tsingke Biotech Co., Ltd., Beijing, China (Figure S1).

2.4. Compatibility Test

Based on the method of Dalvinder et al. [31] with minor modifications, the five dominant fungi, two bacteria and one actinomycete were inoculated onto carboxymethyl cellulose solid culture medium with crossing lines between two strains. The cultures were incubated at 30 °C for 4–5 days to observe any antagonism between the strains during growth (Table S1).

2.5. Construction of Microbial Strain Consortium

2.5.1. Enzyme Activity and Cellulose Degradation Rate of Two-Microbial-Strain Combinations

To select the dominant fungi and bacteria without antagonistic activity, the inoculation ratios of fungi to bacteria (F/B) were set at 1:1 in a liquid fermentation medium for straw degradation, with a control group containing 5% sterile water. Carboxymethyl cellulase, filter paper enzyme and β-glucosidase activities were measured according to the method described by Lu et al. [33].
For the carboxymethyl cellulase (CMCase) assay, the reaction mixture consisted of 0.2 mL of crude enzyme solution and 0.2 mL of 1% sodium carboxymethyl cellulase buffer, incubated at 50 °C for 30 min. The enzyme reaction was then terminated by adding 0.6 mL of 3,5-dinitrosalicylic acid (DNS) solution, followed by boiling in a water bath for 10 min. After cooling to room temperature, a constant volume of deionized water was added to a final volume of 10 mL and mixed thoroughly, and the absorbance was measured at 540 nm [33].
Filter paper enzyme (FPase) activity was determined by mixing 10 mg of filter paper (Whatman No. 1, 0.2 × 1.2 cm) with 0.2 mL of enzyme solution and 0.2 mL of acetic acid–sodium acetate buffer (50 mM, pH 5.0) in a water bath at 50 °C for 60 min. The enzyme reaction was terminated by adding 0.6 mL of DNS solution and boiling for 10 min. After cooling to room temperature, the mixture was diluted to 10 mL with deionized water, mixed thoroughly and allowed to stand for 20 min to enable complete precipitation of the reactants. The absorbance was then measured at 540 nm [33].
The β-glucosidase activity was determined by mixing 0.2 mL of crude enzyme with 0.9 mL of 4-nitrophenyl-β-d-glucopyranoside buffer (pNPG, 5 mM) at 50 °C for 10 min, and the reaction was terminated by adding 2 mL Na2CO3 solution (1 M), followed by cooling to room temperature and mixing well. The absorbance was then measured at 405 nm [33].
The cellulose degradation rate was measured according to the method of Li et al. [13] with minor modifications. Fungi and bacteria combinations were inoculated onto the culture medium and incubated at 28 °C for 30 days with a control group containing 5% sterile water. The degradation rate was determined every 10 days. Finally, five fungi–bacteria combinations were selected for further analysis (Table S2).

2.5.2. Enzyme Activity and Cellulose Degradation Rate of Three-Microbial-Strain Combinations

After determining the degradation rate of the two-microbial-strain combinations, five combinations with the highest degradation rates were selected and further combined with actinomycetes to form three-microbial-strain consortia (Table S3). The inoculation ratios of fungi/bacteria/actinomycetes were set at 1:1:1 in a liquid fermentation medium for straw degradation, with a control group containing 5% sterile water. Carboxymethyl cellulase, filter paper enzyme, β-glucosidase activity and the degradation rate of the three-microbial-strain consortia were measured as described in Section 2.5.1.

2.6. pH Changes of Combined Microbial Strains

The pH value of the decayed corn straw fermentation broth was measured using a pH meter (PHS-3C type pH meter, Shanghai Yi Electrical Scientific Instruments Co., Ltd., Shanghai, China) on the 0th, 10th, 20th and 30th day, respectively.

2.7. Composting Effect of Selected Microbial Strain Combinations for In Situ Fermentation

The composting experiment was conducted in Caoba Village, Mengzi City, Yunnan Province, China. Two composting groups were established: a control group without inoculants and a treatment group with various microbial combinations (A1, A4, A5, B5, B6, B7 and C; B5, B6 and B7 were previously isolated and maintained in our laboratory) (Table 1). A compost pile with a length, width and height of 1.5 m, 1.2 m and 0.7 m was constructed. The inoculant volume of compound microbial inoculants was 0.2% of the dry weight of the composting sample, sprayed evenly over the compost and mixed quickly. The corn straw was cut into pieces of 3–5 cm, and the carbon-to-nitrogen (C/N) ratio of the straw piles was adjusted to 25:1 using urea. The urea was dissolved in water, evenly sprinkled on the surface of the corn straw piles and, thus, the moisture content was adjusted to approximately 65%. Different microbial inoculants were added and mixed 3–4 times. The composting process lasted for 35 days, with the pile being turned and sampled every 7 days. Approximately 50 g samples from the top, middle and bottom of each compost pile were collected, mixed and divided into two parts. One part was air dried for moisture content determination and the other part was refrigerated for pH, E4/E6 value and seed germination index determination. The temperature of the pile was recorded daily at 10:00 a.m. The pH value was measured using a pH meter (PHS-3C type pH meter, Shanghai Yi Electrical Scientific Instruments Co., Ltd., Shanghai, China). The corn straw samples were dried at 105 °C in an oven to determine the moisture content, and the E4/E6 value was measured using a visible ultraviolet spectrophotometer (UV-2100, Shanghai Uniko Chemical Analysis Instrument Co., Ltd., Shanghai, China) by determining the absorbance at 465 nm and 665 nm, respectively [32]. The seed germination index was determined following the method of Zucconi et al. [34].

2.8. Statistical Analysis

All treatments and measurements were investigated with three biological replications. The statistical analyses were conducted by SPSS Statistics software (v20.0) and comparisons between treatments were conducted by Duncan’s test (p < 0.05). All components of the cultural medium are shown in Table S4.

3. Results

3.1. Screening and Identification of Corn-Straw-Degrading Microbial Strains

The microbial strains including 12 fungi, 8 bacteria and 7 actinomycetes were obtained from decayed corn straw through enrichment and primary screening. Then, five fungi strains (labeled as A1, A2, A3, A4, A5), two bacteria strains (labeled as B1 and B2) and one actinomycete strain (labeled as C) were rescreened based on enzyme activity and degradation rate (Figure 1A,B). The size of the transparent circle correlated with enzyme activity, with larger circles indicating higher enzyme activity (Figure 2). The CMCase of strain B1, at 32.06 U/mL, was significantly higher than other single strains, and the FPase of strain A3 at 36.20 U/mL was significantly higher than that of other single strains. Similarly, the β-Gase activity of strain A1 at 35.56 U/mL was significantly different from that of other single strains (p < 0.05) (Figure 1A). The diameter of the transparent circle to the colony of each strain is shown in Table 2. To further clarify the strains, 16S rRNA/ITS sequencing was performed on five fungi strains, two bacteria strains and one actinomycete strain. The final identifications are as follows: A1 is identified as Neurospora intermedia isolate 29, A2 is Streptomyces isolate FFJC33, A3 is Gibberella moniliformis isolate FKCB-009, A4 is Fusarium fujikuroi isolate EFS3(2), A5 is Fusarium Fujikuroi isolate FZ04, B1 is Lysine bacillus macroides strain LNHL43, B2 is Bacillus subtilis strain MPF30 and C is Paenibacilli lautus strain ALEB-P1.

3.2. Enzyme Activity and Degradation Rate of Corn Straw Using Single Strain

The enzyme activities varied significantly among the strains with each strain exhibiting substantial differences in the same enzyme activity (Figure 1A). The CMCase activity of the bacterium strain B1 (32.06 U/mL) was higher than that of fungi and actinomycetes (p < 0.05). For FPase and β-Gase, the fungi strains showed higher activity than the bacteria and actinomycetes. The FPase of fungus A3 (36.20 U/mL) and the β-Gase of fungus A1(35.56 U/mL) were significantly higher than those the other strains (p < 0.05).
As shown in Figure 1B, the degradation rate of corn straw by five fungi, two bacteria and one actinomycete showed gradual increases with the extension of incubation time. All strains demonstrated higher degradation rates compared to the control. The corn straw degradation rate of fungus A4 was significantly higher than that of the other strains (p < 0.05). The degradation rates of fungus A4 were 22.32%, 28.14% and 37.64% on the 10th, 20th and 30th days, respectively, which were 13.14%, 16.02% and 22.35% higher than those of the control.

3.3. Compatibility Test and Enzyme Activity of Combined Microbial Strains

In the screening phase, based on the enzyme activity and degradation rate, five fungi strains and two bacteria strains were selected. The fungi, bacteria and actinomycetes without antagonistic effects were combined (Table S1). The five dominant fungi (A1, A2, A3, A4, A5) without antagonism with two dominant bacteria (B1, B2) were combined to form two-microbial-strain consortia (Table S2). These two-strain combinations (A1B1, A2B2, A3B1, A3B2, A4B2) were further combined with actinomycetes to create three-microbial-strain consortia (Table S3).
The CMCase, FPase and β-Gase activities of different strain combinations are shown in Figure 1C,D. Among the two-strain combinations, the CMCase activity of combination A4B1 (40.64 U/mL) was significantly higher than that of the other combinations (p < 0.05) The FPase activities of combination A2B2 and A3B2 were significantly higher than other combinations (p < 0.05), with FPase values of 55.44 U/mL and 55.02 U/mL, respectively. The β-Gase activities of A2B1 and A2B2 were 26.26 U/mL and 25.73 U/mL, which were significantly higher than those of other strain combinations (p < 0.05). Overall, A2B2 exhibited the highest activity for all three enzymes among the two-microbial-strain combinations (Figure 1C).
Figure 1D shows the enzyme activities in the three-strain combinations. The CMCase activities of A2B2C, A4B2C, A1B1C and A3B2C were significantly higher than that of A3B1C, with values of 35.88 U/mL, 35.03 U/mL, 33.87 U/mL and 33.77 U/mL, respectively. The FPase activity of combination A4B2C (63.59 U/mL) was significantly higher than that of other combinations (p < 0.05). Similarly, the β-Gase of A4B2C (26.15 U/mL) was significantly higher than of the other combinations (p < 0.05). The A4B2C combination had the highest enzyme activity among the three-strain combinations.

3.4. Degradation Rate of Corn Straw Using Combined Microbial Strains

The degradation rate increased gradually with the extension of incubation time, regardless of whether a single strain or strain combinations were used. The degradation rate of all microbial strain treatments was higher than that of the control. After 30 days of fermentation, the A4 strain demonstrated a significantly higher degradation rate of 37.64% compared to the other single strains (p < 0.05) (Figure 1B).
The degradation rate in the strain combination treatments also showed a gradual upward trend over time. All strain combinations showed significant degradation effects during the fermentation period from 0 to 10 days. Among two-microbial-strain combinations, the degradation rates of A2B2 and A1B1 were significantly higher compared to other treatments (p < 0.05), with values of 40.33% and 40.05%, respectively. In contrast, the control had the lowest degradation rate of 13.08% after 30 days of fermentation. The top five strain combinations in terms of degradation rate on the 30th day were A1B1, A2B2, A3B1, A3B2 and A4B2, with rates of 40.05%, 40.33%, 39.85%, 38.78% and 37.10%, respectively (Figure 3A).
For three-strain combinations, the degradation rate of A3B2C was significantly higher than that of other strain combinations on the 10th day (p < 0.05). However, on the 20th and 30th days, A1B1C demonstrated a significantly higher degradation rate, with values of 32.10% and 37.43%, respectively (p < 0.05). The degradation rates of corn straw by other combinations of strains, A2B2C, A3B1C, A3B2C and A4B2C, were 32.83%, 34.90%, 32.23% and 30.63%, respectively, on the 30th day.

3.5. pH Value Changes during the Degradation of Corn Straw by Combined Microbial Strains

The pH value of the fermentation broth for two-strain combinations increased from 6.95 to 7.12, while for the three-strain combinations, it rose from 7.00 to 7.12 (Figure 3C,D). The pH values initially increased and then gradually decreased over time during the fermentation process. At the start of fermentation, the pH values showed an upward trend, which may be due to the production of some alkaline metabolites by the composite microbial strains during the fermentation process. In the later stage of fermentation, the pH values declined as these metabolites were consumed, and the straw decomposed.

3.6. Changes in Physicochemical Properties during In Situ Corn Straw Composting

3.6.1. Temperature Changes with Different Microbial Inoculants

The compost pile temperature under both control and microbial inoculant treatments increased rapidly from day 2 to day 12. The pile temperature increased most rapidly under T7 treatment, reaching above 50 °C on day 4 (Figure 4A). From the 7th day onward, the pile temperature was higher under T7 treatment compared to other microbial inoculants. The inoculation of combined microbial strains accelerated the temperature rise, facilitated the decomposition of organic matter and allowed the compost to reach maturity earlier.

3.6.2. Moisture Content Changes with Different Microbial Inoculants

As shown in Figure 4B, the moisture content of all compost piles remained between 72.57% and 77.13% during the initial stage of fermentation. At the end of composting, the moisture content ranged from 59.95% to 65.15%, with the lowest moisture content (59.95%) under T3 treatment.

3.6.3. pH Changes during with Different Microbial Inoculants

During the composting process, the pH value increased initially and subsequently decreased. The initial pH value of the compost piles increased from 8.64 to 8.72, likely due to the release of ammonium nitrogen from decomposition of organic matter. In the middle stage, from day 7 to day 14, the pH value decreased, likely due to the volatilization of ammonium nitrogen and ongoing nitrification (Figure 4C).

3.6.4. E4/E6 Changes Value with Different Microbial Inoculants

The E4/E6 values changed during composting with microbial inoculants. The initial E4/E6 value increased from 4.11 to 4.65, and by the end of composting, it ranged from 3.02 to 6.19. The lowest E4/E6 value was observed in the T7 treatment, indicating higher maturity and stability compared to the other microbial inoculant treatments. The E4/E6 values under T1, T2, T3, T5 and T7 treatments were lower at the end of composting compared to the initial stage, whereas the values under T4, T6 and control treatments were higher in the initial stages of fermentation (Figure 4D).

3.6.5. Seed Germination Index Changes with Different Microbial Inoculants

In this study, a strong inhibitory effect on seed germination was observed during the initial stage of fermentation, with the seed germination index first decreasing and then increasing (Figure 4E). By the end of composting, the germination index for all piles exceeded 60%. The control had the lowest germination index, while the compost treated with microbial inoculants showed a higher maturity compared to that without inoculant. The germination index of the composts under T2, T4, T6 and T7 treatments reached 80%, while the index under T7 treatment reached 95%, indicating that T7 treatment was beneficial for promoting seed germination (Figure 4E).

4. Discussion

4.1. Identification and Screening of Microbial Strains

In this study, five dominant fungi, two dominant bacteria and one dominant actinomycete were identified using Congo red staining, enzyme activity assays and degradation rate measurements (Figure 2, Table 2). After antagonistic tests, the strains without antagonistic effects were combined into fungal and bacterial combinations (Table S1). From these combinations, strains with better degradation effects were selected and combined with actinomycetes to form a three-strain consortium (Tables S2 and S3). The CMCase activities of both the two-strain and the three-strain combinations were higher than that of a single strain. However, the CMCase activities of the three-strain combinations was lower than that of the two-strain combinations (Figure 1). In both combinations, β-Gase activity was lower than that in a single strain (Figure 1). Similar observations were reported by Li et al. [25], where the CMCase, FPase and β-Gase of some combinations were lower than those of single strains, potentially due to differing metabolic secretions within the microbial consortium. In terms of FPase, both the two-strain combination and three-strain combination exhibited higher activity compared to the single strain, with the three-strain combination showing the highest FPase activity. Mou et al. [26] reported that Trichoderma asperellum LYS1 produced an abundant cellulase–hemicellulase enzyme cocktail for lignocellulosic biomass degradation, especially for hemicellulose-rich biomass. Kaur et al. [35] used a natural variant of Aspergillus niger P-19 for the in-house production of a cellulase–hemicellulase consortium, achieving of 126, 36, 47, 693 and 57 IU/g for CMCase, FPase, β-Gase, xylanase and mannanase, respectively, after 5 days. Raghuwanshi et al. [36] found that a mutant strain of T. asperellum SR1-7 produced filter paper enzyme activity (2.2 IU/gds), carboxymethyl cellulase activity (13.2 IU/gds) and β-glucosidase activity (9.2 IU/gds) under optimized conditions, achieving 1.4-, 1.3- and 1.5-fold higher activities than the wild type. Lepcha and Ghosh [37] reported that a thermophilic microbial consortium produced several extracellular GHs, such as endoglucanase, exoglucanase, β-Gase, endoxylanase and β-xylosidase, with enhanced activity at 60 °C compared to 37 °C and at a pH of 5–6 after 48 h of incubation. Samir Ali et al. [19] screened and constructed of a novel microbial consortium SSA-6 from the gut symbionts of wood-feeding termites, showing significant synergistic cellulolytic, xylanolytic and ligninolytic activities compared to a single strain. Similarly, Han et al. [38] found that a bacterial consortium composed of six strains exhibited a synergistic enzymatic pattern, resulting in a cornstalk weight loss of 43.62%, 15.08% higher than the control. Our findings align with these studies, as the straw degradation efficacy of the composite microbial consortium surpassed that of a single strain (Figure 3A). However, the degradation rates of corn straw by the three-strain combinations were lower than those of the two-strain combinations (Figure 3B). Previous studies have also indicated that microbial consortia accelerate the corn straw degradation more effectively than single strain treatments [39].

4.2. Corn Straw Composting In Situ

Composting is a biochemical process mediated by a variety of microorganisms that destroy pathogens and recycle nutrients. The changes of temperature, pH and moisture are essential indicators for assessing the effectiveness of composting. Specifically, internal temperature reflects the degree of maturity of composting and influences the activity of microbial strains during the composting process [40]. Temperature fluctuations affect pollutant removal efficiency and the degradation of organic fractions in corn straw composting [41,42]. In this study, the temperature inside the pile under T7 treatment was higher than in other treatments from the 7th day (Figure 4A), indicating that the combined microbial inoculants accelerated the temperature rise. Water is essential for microbial growth during composting [43]. Studies have demonstrated that excessively high moisture content can lead to a decrease in compost quality and create anaerobic conditions due to waterlogging [44]. Liu et al. [45] reported that moisture content gradually decreases as fermentation progresses, remaining within the 45% to 60% range. In our study, the moisture content decreased in all piles throughout the composting process (Figure 4B), consistent with previous findings [46]. The pH plays an important role in the growth of microorganisms, with an optimal range of 6.7 to 9.0 during composting [47]. A pH between 8 and 9 improves composting efficiency and helps avoid odor problems caused by delayed reactions [48]. During the middle stage (7 to 14 days), the pH decreased due to ammonium nitrogen volatilization and nitrification (Figure 4C), similar to results reported by Wang et al. [32]. The E4/E6 ratio indicates the degree of condensation and aromatization of humic acid [49]. A lower E4/E6 value signifies greater condensation and humus stability in compost piles; however, an increase in E4/E6 during composting is likely due to the biological decomposition of lignin into small-molecule organic matter in corn straw [50,51]. In this study, E4/E6 values under T1, T2, T3, T5 and T7 treatments were lower at the end of composting than at the beginning (Figure 4D), likely due to the consumption of small organic acids and the conversion of smaller humic acids into large ones [52]. In contrast, E4/E6 values under T4, T6 and the control treatments were higher in the early stages (Figure 4D), possibly due to the degradation of cellulose and hemicellulose into small molecules [22]. Seed germination index (GI) is an indicator of compost maturity and phytotoxicity [53]. In our study, at the end of composting, the germination index of all piles exceeded 60% (Figure 4E), indicating that the compost had matured and its toxicity was within plant tolerance limits [54]. Composting is generally considered mature and non- toxic when the GI exceeds 50%, with full decomposition indicated by a GI above 80% [54]. Numerous studies have demonstrated that adding microbial inoculants can shorten composting time and improve compost quality [20]. Microorganisms have high conversion efficiency, enhancing potential and reliability of microbial agents in composting.

5. Conclusions

In this study, we employed a strategy to develop a microbial strain consortium for the degradation of corn straw. We isolated and screened microorganisms from decayed corn straw, identifying five dominant fungi, two dominant bacteria and one dominant actinomycete based on their efficacy in promoting corn straw weight loss and producing significant quantities of synergistic enzymes. The optimal microbial consortium, A2B2, was a combination of Streptomyces FFJC33 and Bacillus subtilis MPF30. This combination showed significantly higher FPase, β-Gase and degradation rate compared to other microbial strain treatments. In situ composting experiments revealed that microbial inoculants accelerated temperature rise, reduced moisture content and the E4/E6 ratio and increased the germination index of the piles. These findings suggest that the microbial consortium had a promoting effect on the fermentation of corn straw. The microbial consortium comprised Neurospora intermediate isolate 29, Fusarium Fujikuroi isolate EFS3(2), Fusarium Fujikuroi isolate FZ04, Lysinibacillus macroides, Lysinibacillus sphaericus, Bacillus subtilis and Paenibacillus lautus, which have the optimum degradation effects on corn straw composting.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14092107/s1, Figure S1: A multilocus phylogenetic analysis based on ITS, 16S rRNA genes with Kimura 2-parameter model using MEGA7.0; Table S1: Compatibility test of different microbial strains; Table S2: Combinations of fungi and bacteria; Table S3: Combinations of fungi, bacteria and actinomycetes; Table S4: Culture medium.

Author Contributions

J.L. (Jie Li), R.Y. and J.L. (Juan Li): Investigation, Methodology, Data curation, Writing—original draft. H.F. and Y.Y.: Data curation, Formal analysis. P.Y.: Investigation, Methodology. C.L.: Supervision, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the project of Yunnan Young and Middle Aged Academic and Technical Leaders Reserve Talents (202205AC160056); Yunnan Fundamental Research Projects (202401AT070059); the Fundamental Research Funds of China West Normal University (19E046).

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 author/s.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 02107 g001
Figure 1. (A) Enzyme activity of single strains. (B) Degradation rate of corn straw using single strain. (C) Enzyme activity of combination of two microbial strains. (D) Enzyme activity of combination of three microbial strains. Different lowercase letters indicate significant differences between different treatments within the same enzyme.
Figure 1. (A) Enzyme activity of single strains. (B) Degradation rate of corn straw using single strain. (C) Enzyme activity of combination of two microbial strains. (D) Enzyme activity of combination of three microbial strains. Different lowercase letters indicate significant differences between different treatments within the same enzyme.
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Figure 2. Hydrolytic circles of fungi, bacteria and actinomycetes.
Figure 2. Hydrolytic circles of fungi, bacteria and actinomycetes.
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Figure 3. (A) Degradation rate of corn straw using two-microbial-strain combinations. (B) Degradation rate of corn straw using three-microbial-strain combinations. (C) pH changes of corn straw using two-microbial-strain combinations. (D) pH changes of corn straw using three-microbial-strain combinations.
Figure 3. (A) Degradation rate of corn straw using two-microbial-strain combinations. (B) Degradation rate of corn straw using three-microbial-strain combinations. (C) pH changes of corn straw using two-microbial-strain combinations. (D) pH changes of corn straw using three-microbial-strain combinations.
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Figure 4. (A) Variation of pile and ambient temperature. (B) Moisture content variation of the pile. (C) pH value variation of the pile. (D) E4/E6 value variation of the pile. (E) Seed germination index variation of the pile.
Figure 4. (A) Variation of pile and ambient temperature. (B) Moisture content variation of the pile. (C) pH value variation of the pile. (D) E4/E6 value variation of the pile. (E) Seed germination index variation of the pile.
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Table 1. Inoculant combination of each treatment.
Table 1. Inoculant combination of each treatment.
InoculantMicrobial Strains
FungiBacteriaActinomycetes
1 (T1)A1 + A4 + A5B5 + B6C
2 (T2)A1 + A4 + A5B5 + B7C
3 (T3)A1 + A4 + A5B6 + B7C
4 (T4)A1 + A4B5 + B6 + B7C
5 (T5)A1 + A5B5 + B6 + B7C
6 (T6)A4 + A5B5 + B6 + B7C
7 (T7)A1 + A4 + A5B5 + B6 + B7C
CKDistilled water
A1: Neurospora intermedia isolate 29, A4: Fusarium fujikuroi isolate EFS3(2), A5: Fusarium fujikuroi isolate FZ04, B5: Lysinibacillus macrolides, B6: Lysinibacillus sphaericus, B7: Bacillus subtilis, C: Paenibacillus lautus.
Table 2. The diameter ratio value of the transparent circle to the corresponding colony.
Table 2. The diameter ratio value of the transparent circle to the corresponding colony.
Strain LabelD/d (cm)
A15.95 ± 0.63 bc
A25.89 ± 0.70 bc
A37.37 ± 1.16 abc
A45.06 ± 1.93 c
A56.78 ± 1.07 abc
B13.11 ± 0.12 b
B23.20 ± 0.12 b
C2.60 ± 0.06 b
Different lowercase letters in the same column indicate significant differences in the diameter of the transparent circle between different single strains.
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Li, J.; Li, J.; Yang, R.; Yang, P.; Fu, H.; Yang, Y.; Liu, C. Construction of Microbial Consortium to Enhance Cellulose Degradation in Corn Straw during Composting. Agronomy 2024, 14, 2107. https://doi.org/10.3390/agronomy14092107

AMA Style

Li J, Li J, Yang R, Yang P, Fu H, Yang Y, Liu C. Construction of Microbial Consortium to Enhance Cellulose Degradation in Corn Straw during Composting. Agronomy. 2024; 14(9):2107. https://doi.org/10.3390/agronomy14092107

Chicago/Turabian Style

Li, Jie, Juan Li, Ruopeng Yang, Ping Yang, Hongbo Fu, Yongchao Yang, and Chaowei Liu. 2024. "Construction of Microbial Consortium to Enhance Cellulose Degradation in Corn Straw during Composting" Agronomy 14, no. 9: 2107. https://doi.org/10.3390/agronomy14092107

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