Study on the degradation conditions of corn stalks by Asian corn borer digestive enzymes combined with white-rot fungus

Abstract

Since the environmentally friendly reuse of corn stalks attracts more and more attention, it is an efficient and feasible way to reuse corn stalks as forage. However, whether the cellulose, lignin, and hemicellulose within corn stalks can be effectively decomposed becomes a key to reusing corn stalks as forage. Orthogonal test was designed by five different degradation temperatures (22°C, 24°C, 26°C, 28°C, 30°C), five different pH values (4, 5, 6, 8, 10), and five different degradation time durations (5, 15, 25, 30, and 35 days) to examine 25 kinds of different degradation conditions. It was found that the decomposition effect of hemicellulose, cellulose, and lignin, of group 25 (26°C, pH = 5, 25 days) was stronger compared with other groups, with the contents calculated as 8.22%, 31.55%, and 22.55% individually (p < 0.01, p < 0.05). Group 19 (22°C, pH = 4, 5 days) revealed the worst degradation effect of cellulose, lignin, and hemicellulose compared to other groups, with contents calculated as 15.48%, 38.85%, and 29.57%, individually (p < 0.01, p < 0.05). The research data deliver a basis for ideal degradation conditions for corn stalks degradation in combination with the digestive enzymes of P. chrysosporium and O. furnacalis larva. Aiming to explore a highly efficient and environmentally friendly corn stalk degradation method.

Keywords

Corn stalks degradation digestive enzymes lignocellulose white-rot fungi

Introduction

Corn stalks can be employed as a reusable resource that can bring huge economic benefits. The nutrition content of corn stalks contains more than 20% carbohydrates, 2%–4% protein, and 0.5%–1% fat.¹ The composition of corn stalks is lignin, cellulose, hemicellulose, and ash, but lignin, cellulose, and hemicellulose are the main components of corn stalks, of which lignin is the most difficult to degrade.¹ Therefore, the difficult degradation of lignin restricts the reuse of corn stalks.^2,3 Using corn stalks as forage for herbivorous animals is one way to reuse them. However, corn stalks contain a lot of lignin, cellulose, and hemicellulose, which cause poor palatability.⁴ In order to reuse corn stalks as forage, it is necessary to effectively degrade the lignin, hemicellulose, and cellulose.

At present, the decomposition of cellulose, lignin, and hemicellulose mainly involves chemical, physical, and biological methods.^5–7 Chemical methods degrade cellulose, lignin, and hemicellulose mainly by acid–base treatment. Although the chemical method can achieve an ideal degradation effect for lignin, cellulose, and hemicellulose, it will cause environmental pollution and chemical residues that it is not suitable for corn stalk reuse as forage. Physical methods change physical properties to degrade lignin, cellulose, and hemicellulose by applying high temperature and high pressure. Though physical methods do not bring significant environmental hazards, the degradation effect is limited and cannot meet the requirements of corn stalk reuse sometimes. Biological degradation of the cellulose, lignin, and hemicellulose by bacteria or fungi. Many studies have manifested that fungi are effective in the decomposition of cellulose, lignin, and hemicellulose due to their high metabolic versatility and significant involvement in the decomposition of organic matter in soil.^8–11 The advantage of biodegradation is that it is not only environmentally friendly but also possesses high degradation efficiency.¹² Biological methods are regarded as an ideal degradation choice at present.¹³

The influence of environmental factors and varied degradation conditions affect disease progression and insect behavior.^14,15 Currently, white-rot fungi (Phanerochaete chrysosporium) are considered a kind of microorganism that can break through the degradation barrier of refractory lignin in lignocellulose structure and play an indispensable role in the biological organic carbon cycle.¹⁶ Wei et al.¹⁷ confirmed that six stains of white-rot fungus could degrade stalks with P. chrysosporium being the most effective in degrading cellulose and hemicellulose with degradation rates of 42% and 30%, respectively. Zeng et al.⁴ investigated that white-rot fungus are able to degrade the lignin of corn stalks, with the average degradation of lignin reaching 33.4%, and also found that degrading lignin in combination with white-rot fungus and urea could increase the content of protein of corn stalks by 180% that was suitable for reuse as forage. The degradation of lignocellulose by white-rot fungi is synergistic with many enzyme systems (Figure 1). Bak¹⁸ proved that there were 39 metabolites and 123 regulatory genes involved in lignocellulosic hydrolysis, enabling the degradation process through intracellular regulatory networks and cascade reactions. Whereas the degradation conditions, such as temperature, pH, time, and so on, could affect the metabolic ability of white-rot fungi to produce enzymes, thus affecting the degradation effect.^19,20 Wang et al.²¹ explored that the degradation impact of white-rot fungi could be affected by short-term pre-exposure to silver ions.

Figure 1.

Enzyme system synergistic degradation process.

Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), is a destructive agricultural pest that carves corn stalks, mainly in Asian countries (Figure 2).²² Insect digestive enzymes mainly include amylase, protease, lipase, and so on.^23–25 Corn stalks eaten by O. furnacalis larva will be digested and decomposed by digestive enzymes of the midgut to break down cellulose, lignin, and hemicellulose for better nutrient absorption.²⁶ Wang et al.² found that digestive enzymes of the O. furnacalis larva can significantly improve degradation by white-rot fungus (P. chrysosporium) in cellulose, lignin, and hemicellulose of corn stalks. Consequently, the digestive enzymes of O. furnacalis larva exhibit great potential to be utilized as synergists.

Figure 2.

The Asian corn borer (Ostrinia furnacalis) larva.

Based on our previous study, the influence degree of different degradation time durations, different degradation pH values, and different degradation temperatures on the degradation effect was studied in this research, aiming to explore an optimal degradation condition of the combination of digestive enzymes of P. chrysosporium and O. furnacalis larva.

Materials and methods

Corn stalk preparation

Corn stalks harvested after the autumn harvest from farmlands located in Lianyungang City, Jiangsu province, China. Corn stalks were crushed into 18 mesh, dried to constant weight at 60°C, and sterilized at 121°C for 20 min before the experiment. In our research, the base components of corn stalks included ash (5.7 ± 1.41%); hemicellulose (29.65 ± 0.13%); cellulose (38.9 ± 0.69%); moisture (8.8 ± 1.28%) and lignin (15.51 ± 0.47%).

White-rot fungus preparation

The purity of white-rot fungus which was obtained from the USA and manufactured by BeNa Culture Collection Company, Beijing, China was verified at Henan Industrial Microbiology Engineering Research Center, Henan, China. The white-rot fungus cultured on potato dextrose agar (PDA) solid culture medium which was prepared by dissolving glucose 20.0 g, KH₂PO₄ 3.0 g, MgSO₄·7H₂O 1.5 g, VB1 1 mg, agar 20 g in 1000 ml potato filtrate and liquid culture medium which was prepared by dissolving glucose 20.0 g, MgSO₄·7H₂O 1.5 g, KH₂PO₄ 3.0 g, VB1 1 mg in 1000 ml potato filtrate at 28°C temperature for 7 days.2 The pH of PDA solid and lipid culture medium both were 6.0 ± 0.2.

O. furnacalis larvae digestive enzyme solution preparation

The insect sample, O. furnacalis larvae, was obtained from Nongan City, Jilin Province, China. and preserved in the Provincial Key Laboratory of Materials, Jilin Engineering Normal University. The larvae were cultured at 26°C ± 2°C temperature, 70% ± 5% relative humidity, L15:D9 photoperiodic conditions, and fed artificially.²⁷ In this study, healthful O. furnacalis larvae were utilized to extract digestive enzymes. After rinsing O. furnacalis larvae with distilled water, impalement of gastropods to drain hemolymph, and the dorsal side of larvae was dissected to remove the midgut. Then, 0.1 ml of 0.15 mol/L NaCl solution was added. and O. furnacalis larval digestive enzyme solution was obtained after homogenizing the mixture (4°C) and centrifuging at 15,000 rpm (10 min) for supernatant. The larval digestive enzyme activity was determined according to the assay kits of amylase lipase, and trypsin, respectively (Code: C016-1-1, Code: A054-1-1, Code: A080-2-2, Jiancheng, Nanjing).²

Decomposition of corn stalks by Asian corn. borer digestive enzymes combined with white-rot fungus under different condition

Five different degradation temperatures (22°C, 24°C, 26°C, 28°C, 30°C), five different pH gradients (4, 5, 6, 8, 10), and five different degradation time durations (5, 15, 25, 30, and 35 days) were selected to design orthogonal table for the experiment with the aid of SPSS Statistics 25 for Mac (SPSS Inc., Chicago, USA). A L₂₅(5³) orthogonal table was generated (Table 1).

Table 1.

Orthogonal table.

Group number	Temperature (°C)	pH	Day (d)
1	22	6	25
2	22	5	15
3	24	6	5
4	26	4	15
5	28	6	15
6	26	10	5
7	26	6	30
8	28	8	25
9	30	8	5
10	24	8	15
11	26	8	35
12	28	4	35
13	30	5	30
14	30	6	35
15	24	10	25
16	30	10	15
17	22	8	30
18	22	10	35
19	22	4	5
20	28	10	30
21	30	4	25
22	24	5	35
23	26	5	25
24	28	5	5
25	24	4	30

Our previous study found that the addition of 9 ml of O. furnacalis larval digestive enzyme solution to the sample with sterilized corn stalks (30 g) and white-rot fungus suspension (90 ml) appeared a significant degradation effect. 9 ml solution of O. furnacalis larval digestive enzyme, disinfected corn stalks (30 g), and white-rot fungus suspension (90 ml) were mixed into 250 ml conical bottle as each sample. The samples were then placed in corresponding conditions mentioned in Table 1 for the degradation test. Each sample was analyzed three times for cellulose, hemicellulose, or lignin content. VanSoest method and ANKOM-2000 lignocellulose analyzer from the USA were performed to determine the components of each sample.²⁸

Statistical analysis

Apple Numbers (Apple Co., California, USA) were used to calculate the standard deviation (SD) and mean value (MV) of content percentages of hemicellulose, cellulose, and lignin. Experimental data were also classified and recorded using Apple Numbers (Apple Co., California, USA). One-way analysis of variance was determined according to SPSS Statistics 25 for Mac (IBM, Chicago, USA). p = 0.05 and p = 0.01 were set as significant differences.

Results

Degradation of lignin

The results are shown in Table 2, with varied lignin contents for each test group. Group 23 (26°C, pH = 5, 25 days) appeared the lowest lignin content compared with other groups, which was 8.22% (p < 0.01, p < 0.05). However, the lignin concentration of group 22 (24°C, pH = 5, 35 days) was slightly higher than that of group 23, which was 9.43% (p < 0.05, p < 0.01). Group 19 (22°C, pH = 4, 5 days) indicated the highest lignin content compared with other groups, which was 15.48% (p < 0.01, p < 0.05).

Table 2.

Orthogonal test of lignin degradation.

				Lignin content (%)	Significance of difference
Group number	Temperature (°C)	pH	Day (d)	MV ± SD	0.05	0.01
1	22	6	25	14.71 ± 0.07	E	d
2	22	5	15	14.81 ± 0.02	D	d
3	24	6	5	15.44 ± 0.04	B	b
4	26	4	15	9.75 ± 0.02	S	r
5	28	6	15	11.83 ± 0.03	M	l
6	26	10	5	15.34 ± 0.01	B	b
7	26	6	30	10.94 ± 0.01	P	o
8	28	8	25	13.45 ± 0.02	I	h
9	30	8	5	15.12 ± 0.02	C	c
10	24	8	15	12.63 ± 0.03	K	j
11	26	8	35	12.54 ± 0.04	K	j
12	28	4	35	14.58 ± 0.01	F	e
13	30	5	30	12.63 ± 0.03	K	j
14	30	6	35	10.67 ± 0.03	Q	p
15	24	10	25	12.29 ± 0.01	L	k
16	30	10	15	10.27 ± 0.01	R	q
17	22	8	30	10.97 ± 0.03	P	o
18	22	10	35	11.59 ± 0.01	N	m
19	22	4	5	15.48 ± 0.01	A	a
20	28	10	30	11.41 ± 0.02	O	n
21	30	4	25	14.27 ± 0.09	G	f
22	24	5	35	9.43 ± 0.19	T	s
23	26	5	25	8.22 ± 0.09	U	t
24	28	5	5	13.93 ± 0.01	H	g
25	24	4	30	13.29 ± 0.12	J	i

Note. Capital letters indicate a very significant difference (p < 0.01); lowercase letters indicate a significance (p < 0.05).

Degradation of cellulose

The results are organized in Table 3, with varied cellulose contents of each test group. Group 23 (26°C, pH = 5, 25 days) appeared the lowest cellulose content compared with other groups, which was 31.55% (p < 0.01, p < 0.05). The cellulose contents of group 4 (26°C, pH = 4, 15 days) and group 22 (22°C, pH = 5, 35 days) were slightly higher than that of group 23, which were 32.03% and 32.26% individually (p < 0.05, p < 0.01). Group 19 (22°C, pH = 4, 5 days) appeared the highest cellulose content by contrast with other groups, which was 38.85% (p < 0.05, p < 0.01).

Table 3.

Orthogonal test of cellulose degradation.

				Cellulose content (%)	Significance of difference
Group number	Temperature (°C)	pH	Day (d)	MV ± SD	0.05	0.01
1	22	6	25	36.49 ± 0.07	DE	de
2	22	5	15	36.68 ± 0.07	D	d
3	24	6	5	37.74 ± 0.13	B	b
4	26	4	15	32.03 ± 0.41	R	p
5	28	6	15	33.84 ± 0.06	K	ij
6	26	10	5	37.4 ± 0.19	C	bc
7	26	6	30	32.85 ± 0.06	NO	mn
8	28	8	25	35.21 ± 0.11	GH	f
9	30	8	5	37.14 ± 0.06	C	c
10	24	8	15	34.43 ± 0.1	IJ	gh
11	26	8	35	34.26 ± 0.06	J	h
12	28	4	35	36.29 ± 0.07	EF	e
13	30	5	30	34.7 ± 0.06	I	g
14	30	6	35	32.69 ± 0.05	OP	mn
15	24	10	25	34.18 ± 0.06	J	hi
16	30	10	15	32.47 ± 0.06	PQ	no
17	22	8	30	33.03 ± 0.06	MN	lm
18	22	10	35	33.51 ± 0.05	L	jk
19	22	4	5	38.85 ± 0.51	A	a
20	28	10	30	33.35 ± 0.11	LM	kl
21	30	4	25	36.15 ± 0.05	F	e
22	24	5	35	32.26 ± 0.18	QR	op
23	26	5	25	31.55 ± 0.28	S	q
24	28	5	5	35.47 ± 0.06	G	f
25	24	4	30	35.18 ± 0.06	H	f

Note. Capital letters indicate a very significant difference (p < 0.01); lowercase letters indicate a significance (p < 0.05).

Degradation of hemicellulose

Results with varied hemicellulose contents of each test group are displayed in Table 4. Group 23 (26°C, pH = 5, 25 days) appeared the lowest cellulose content compared with other groups, which was 22.55% (p < 0.01, p < 0.05). Besides, the hemicellulose concentration of group 22 (24°C, pH = 5, 35 days) was slightly higher than that of group 23, which was 23.07% (p < 0.05). Group 19 (22°C, pH = 4, 5 days) exhibited the highest hemicellulose content by contrast with other groups, which was 29.57% (p < 0.05, p < 0.01).

Table 4.

Orthogonal test of hemicellulose degradation.

				Hemicellulose content (%)	Significance of difference
Group number	Temperature (°C)	pH	Day (d)	MV ± SD	0.05	0.01
1	22	6	25	27.51 ± 0.05	F	ef
2	22	5	15	27.7 ± 0.07	E	e
3	24	6	5	29.21 ± 0.1	B	b
4	26	4	15	23.22 ± 0.19	T	q
5	28	6	15	24.51 ± 0.06	P	l
6	26	10	5	28.65 ± 0.02	C	c
7	26	6	30	23.7 ± 0.05	R	no
8	28	8	25	26.52 ± 0.06	J	h
9	30	8	5	28.18 ± 0.12	D	d
10	24	8	15	25.58 ± 0.06	M	j
11	26	8	35	25.29 ± 0.07	N	k
12	28	4	35	27.34 ± 0.02	G	f
13	30	5	30	25.73 ± 0.06	L	jo
14	30	6	35	23.52 ± 0.17	S	p
15	24	10	25	25.14 ± 0.06	O	k
16	30	10	15	23.48 ± 0.07	S	p
17	22	8	30	23.81 ± 0.07	R	n
18	22	10	35	24.29 ± 0.17	Q	m
19	22	4	5	29.57 ± 0.03	A	a
20	28	10	30	24.21 ± 0.1	Q	m
21	30	4	25	27.11 ± 0.01	H	g
22	24	5	35	23.07 ± 0.06	U	q
23	26	5	25	22.55 ± 0.01	V	r
24	28	5	5	26.67 ± 0.11	I	h
25	24	4	30	26.18 ± 0.06	K	i

Note. Capital letters indicate a very significant difference (p < 0.01); lowercase letters indicate a significance (p < 0.05).

Discussion

The degradation conditions can notably influence the degradation effect of fungi. Degradation temperature is a factor that affects the degradation effect of fungi. Microbial degradation of cellulose, lignin, and hemicellulose within corn stalks is a process in which extracellular oxidase secreted by microorganisms leads to the break of chemical bonds within cellulose, lignin, and hemicellulose.²⁹ Laccase and peroxide are the main extracellular oxidases to degrade cellulose, lignin, and hemicellulose and temperature can affect the secretion of these two extracellular oxidases, thus influencing the decomposition effect of cellulose, lignin, and hemicellulose.^30–32 Group 23 achieved the best degradation effect in lignin, cellulose, and hemicellulose. The reason might be that this degradation temperature was more suitable for the growth of P. chrysosporium which could secrete enough extracellular oxidase for degradation and also keep the activity of digestive enzymes of O. furnacalis larva.³³ Wang et al.²⁸ found that explored that Bacillus cereus was efficient in degrading rice stalks at low temperatures. Whereas Wang et al.²⁸ found that P. chrysosporium appeared efficient for degrading corn stalks at relatively high temperatures. Liu discovered that white-rot fungus indicated the best degradation effect in lignin at 30°C. Li et al.³⁰ and Zhang et al.³⁴ found that the activity of digestive enzymes could be significantly affected by the surrounding temperature. So, the surrounding temperature could affect the degradation effect.

pH is one of the factors affecting the degradation effect. pH not only affects the dissociation and microstructure of charged groups on the cell surface but also can influence the absorption of nutrients and the secretion of metabolites.³⁵ Inappropriate pH values tend to change the metabolic pathway of bacteria, resulting in changes in the composition and quantity of metabolites.³³ Group 19 appeared the worst decomposition effect of cellulose, lignin, and hemicellulose. The reason might be the pH affects the P. chrysosporium absorbing nutrients for growth and blocking the secretion of metabolites that weaken the degradation effect. Zhang et al.³⁶ examined that pH could significantly affect the effect of decomposing corn stalks by fungi. Huang et al.³⁷ also investigated that pH could affect the fungi degradation effect. Furthermore, it was also found that the degradation effect varied at the same pH, which might be because degradation temperature and degradation time were different, influencing the degradation effect.^32,33

The degradation time also affects the degradation effect. Group 23 appeared the best decomposition effect on cellulose, lignin, and hemicellulose in only 25 days. Whereas Wang et al.² discovered that the decomposition effect of cellulose, lignin, and hemicellulose increased with degradation time, with the best decomposition effect occurring at 35 days. The reason might be the degradation condition is different that affected degradation time. Also, group 19 achieved the worst decomposition effect about cellulose, lignin, and hemicellulose. The reason might be that 5 days was not enough for the growth of P. chrysosporium.

In our research, the influences of degradation time, temperature, and pH on the decomposition of selected P. chrysosporium in combination with the O. furnacalis larval digestive enzymes were studied and the optimal degradation conditions were obtained. However, many other factors such as humidity, types of digestive enzymes, fungi culture media, and so on, which affect the degradation effect still need to be further investigated.

Conclusions

Using corn stalks as forage is an important method to reuse corn stalks environmentally friendly. However, the decomposition degree of lignocellulose within corn stalks is the key to judging whether the resulting forage is palatable. The Present study found that the degradation condition of group 25 (26°C, pH = 5, 25 days) was more suitable for the decomposition of corn stalks. This study provides a more suitable degradation condition and method for the farmers to degrade corn stalks after harvest, which could replace incineration and reduce air pollution. The farmers could also try to use the results of this study to degrade and reuse corn stalks as forage to feed livestock, which could reduce the cost. Additionally, the study makes more effective biodegradation of corn stalks for feed companies become possible.

Footnotes

Declaration of conflicting interests

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Department of Education of Jilin Province (JJKH20230238KJ), Science and Technology Development Plan Project of Jilin Province, China (YDZJ202301ZYTS286) and Jilin Engineering Normal University Research Fund (BSKJ202203).

ORCID iD

Yanchen Wang

Author biography

Yanchen Wang major in entomology and bio-degradation. He graduated from Northeast Forestry University. He is working at the Jilin Engineering Normal University. His research focus on entomology, bio-degradation, bio-control and bionic.

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Group number	Temperature (°C)	pH	Day (d)
1	22	6	25
2	22	5	15
3	24	6	5
4	26	4	15
5	28	6	15
6	26	10	5
7	26	6	30
8	28	8	25
9	30	8	5
10	24	8	15
11	26	8	35
12	28	4	35
13	30	5	30
14	30	6	35
15	24	10	25
16	30	10	15
17	22	8	30
18	22	10	35
19	22	4	5
20	28	10	30
21	30	4	25
22	24	5	35
23	26	5	25
24	28	5	5
25	24	4	30

Group number	Temperature (°C)	pH	Day (d)
1	22	6	25
2	22	5	15
3	24	6	5
4	26	4	15
5	28	6	15
6	26	10	5
7	26	6	30
8	28	8	25
9	30	8	5
10	24	8	15
11	26	8	35
12	28	4	35
13	30	5	30
14	30	6	35
15	24	10	25
16	30	10	15
17	22	8	30
18	22	10	35
19	22	4	5
20	28	10	30
21	30	4	25
22	24	5	35
23	26	5	25
24	28	5	5
25	24	4	30

Group number	Temperature (°C)	pH	Day (d)
1	22	6	25
2	22	5	15
3	24	6	5
4	26	4	15
5	28	6	15
6	26	10	5
7	26	6	30
8	28	8	25
9	30	8	5
10	24	8	15
11	26	8	35
12	28	4	35
13	30	5	30
14	30	6	35
15	24	10	25
16	30	10	15
17	22	8	30
18	22	10	35
19	22	4	5
20	28	10	30
21	30	4	25
22	24	5	35
23	26	5	25
24	28	5	5
25	24	4	30