Use Of Spent Mushroom Substrate

Published Date: 02 Nov 2017

Songqing Wu1, Yanjiao Lan1, Yan Peng1, Zhipeng Huang1, Lei Xu1, Ivan Gelbi?2, Xiong Guan1, Shuangquan Zou1*, Lingling Zhang1,3*

1 Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, 350002, People��s Republic of China

2 Biological Centre of the Academy of Sciences of the Czech Republic, Institute of Entomology, Brani?ovsk�� 31, 37005, Czech Republic

3 National Engineering Research Center of Juncao Technology, Fuzhou, Fujian 350002, People��s Republic of China

Songqing Wu and Yanjiao Lan contributed equally to this work.

*Corresponding author. Tel.: +86-591-8378-9259; Fax: +86-591-8378-9259.

E-mail address: [email protected]

Abstract:

The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As locally available agro-industrial by-products, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were detected as following: temperature 32��, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3 and inoculum size 0.5ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1487 ITU/mg) and strong larvicidal persistence of the product were found in the study, which illustrated that SMS based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides.

Keywords

Bacillus thuringiensis; Spent mushroom substrate; Solid-state fermentation; Media

1. Introduction

As an insect vector, mosquito can transfer and cause many diseases, such as malaria, yellow fever, Japanese encephalitis and dengue fever ((Poopathi & Abidha, 2010; Poopathi & Archana, 2012). Worldwide use of chemical insecticides for mosquito control has caused many problems including environmental pollution, insecticide resistance and influence in human health (Bravo et al., 2011; El-Bendary, 2006). As an alternative way to control insect pests, biopesticides, especially derived from Bacillus thuringiensis (Bt), draw more and more attentions in recent years (Prabakaran & Balaraman, 2006; Tokcaer et al., 2006; Zhuang et al., 2011). The larvicidal effect of Bt preparation is based on endotoxin proteins (Cry and Cyt toxins), which were accumulated as crystal inclusions produced upon sporulation (Bravo et al., 2011). Bt-based biopesticides have the advantages of high selectivity and low risk for development of insect resistance (Prabakaran & Balaraman, 2006).

However, the high cost of Bt production medium is the main obstacle for their commercial application to a great extent. Studies indicated that Bt could growth well in medium at a nitrogen content of 0.075%-0.225%, sugar content of 0.1%-1.5% and carbon to nitrogen ratio of 0.44-20.0%. Several agricultural or industrial by-products for producing Bt had been reported, such as coffee husk waste, broiler litter, chicken feathers, wastewater and wastewater sludge (Adams et al., 1999; Adjalle et al., 2009; Poopathi & Abidha, 2008; Poopathi & Abidha, 2011). To date, however, reports on use of spent mushroom substrate (SMS) for Bt cultivation have not been found yet.

In China, approximately 50 million tons of SMS were produced per year (Zhang et al., 2012a). More and more attentions are paid to the disposure and reuse of SMS due to the problem of economic waste and environmental pollution. For this reason, many potential applications of SMS have been developed. Jordan et al. (2008) studied SMS for its ability to improve soil structure and increase dry matter in grassland soils. SMS was also used as growing media for nursery seedlings (Zhang et al., 2012b), biomass resource for ethanol production (Asada et al., 2011; Lee et al., 2008), animal feed (Zhang et al., 1995) and microorganism cultivation (Qiao et al., 2011; Zhu et al., 2012). As for producing Bt by solid-state fermentation, SMS requires less capital investment and moderate pretreated technology (Zhuang et al., 2011).

The aim of this study was to investigate the potential for SMS to be a raw material for mass production of Bt-based biopesticides by solid-state fermentation. Four SMS based media were designed. Spore count, toxicity and other indexes were determined to assess the influences of compositions and conditions of medium for Bt cultivation. The possibility of SMS for Bt commercial production was determined by scale-up fermentation at the optimal condition.

2. Materials and methods

2.1. Bacterial strains

B. thuringiensis subsp. israelensis (Bti) IPS82 used in this study was acquired from the Bacillus Genetic Stock Center (BGSC), Ohio State University, USA. Bti LLP29 was isolated from leaves of Magnolia denudata (Zhang et al., 2010; Zhang et al., 2011). Strains were subcultured and streaked on Luria-Bertani (LB) plates, which contained (g/L): agar 25, tryptone 10.0, NaCl 10.0, and yeast extract 5.0, incubated at 30�� for 12 h and then stored at 4��.

2.2. SMS

The SMS was obtained from Mycological Research Center of Fujian Agriculture and Forestry University. The mushroom substrate that used for cultivating velvet shank, Flammulina velutipes (Curt. ex Fr.) Singer consists of wheat bran, cotton seed hulls, sawdust, lime and light calcium carbonate. After air drying, the SMS was milled to pass 40 mesh screen (425 ��m) and then stored in plastic bags at 4��.

2.3. Analysis of SMS composition and medium

The amount of total sugar and reducing sugar was determined using 3,5-dinitrosalicylic acid reagent (DNS method) (Miller, 1959; Tian et al., 2009) (û��2�������). Organic material was determined according to NY 525-2010. Dissolved organic carbon (DOC) and microbial biomass carbon (MBC) were determined by Shimadzu TOC-VCPH according to Lu (2000). Pretreatments of nitrate nitrogen and ammonia nitrogen were conducted according to the method of Lu (2000). Total nitrogen, nitrate nitrogen and ammonia nitrogen in the steep liquor were determined by continuous flow analyzer (San++, SKALAR, Holland)

2.4. Production of Bt in different media

Four combinations (T1-T4) of SMS based media were designed for Bt production. The T1 combination was composed of 10 g SMS, 0.3g calcium carbonate and 25 ml distilled water. The T2 combination was composed of the T1 mixture and 4g wheat bran. The T3 combination was composed of T2 combination and 0.2g yeast extract. And the T4 combination was composed of T3 combination and 1% inorganic salts (stock solution: 2.03 g/l MgCl2, 1.02 g/l CaCl2 and 0.1 g/l MnCl2). All media compositions except SMS were dissolved in distilled water and the pH was adjusted to 7.2. Each solution added to a 250 ml Erlenmeyer flask containing 10g of SMS. Three replicates for each media were carried out. The flasks were sterilized at 15 psi for 25 min.

2.5. Inoculum and culture conditions

Seed inocula of strains IPS82 and LLP29 were prepared according to Yezza et al. (2006). LB medium (5 ml) was inoculated by a loopful of Bti from LB plate and then grew at 30�� for 12 h as pre-culture. T1-T4 media were inoculated with 10% (v/v) seed culture, respectively. The flasks were subsequently incubated at 150 rpm and 30�� for about 3-4 days. After 90% sporulation, the products were dried in an oven at 55�� for 12 h. Subsequently, the dry media were powdered and stored at 4�� for further use.

2.6. Cell and spore counts

After fermentation, 1g of fermented substrate from T1-T4 media were respectively placed in 250mL Erlenmeyer flasks, to which distilled water and glass bead was added. The culture samples were stored at 4�� after shaking for 4 h.

To determine the viable cell count and spore count, the samples were diluted by 107-1012 times. For the spore count, the diluted samples were subjected to water bath treatment at 75�� for 15 min before plating. After incubating for 24 h, the fully developed colonies between 20 and 300 were counted. Standard deviations for cell count and spore count were 6-8%. Each experiment was performed in triplicate.

2.7. Bioassay

Toxicity was measured by bioassay using Culex quinquefasciatus larvae in fourth����Ч���DZ��õ���2-3�䡣��һ��Ҳ�У��� instar, provided by our owe laboratory, as per WHO procedures (WHO, 2007). The samples (described at 2.6.3) were prepared in appropriate dosages containing 30 larvae each. Three repetitions were performed for each toxin dose at 28��. After 24h, the mortality of each sample was determined thrice on different days by comparing the ?nal mortality of sample preparations with that of sterilized control. The test was effective when larval mortality of the sterile control was less than 10%. Toxicity of sample preparations was determined as LC50 value (mg/L).

2.8. Optimization of the fermentation conditions

The larvicidal effect was determined by bioassays to optimize the fermentation conditions. The effects of temperature and pH values on Bt toxicity were conducted at different temperature (26��-34��) and pH values (pH 5-10). As for moisture content, SMS in five different water percentages (50%- 90%) was added to flask. The effects of inoculum concentration (0.25-1.25ml) and ratio of unpowered to powered SMS (3:1-1:3) were performed respectively. Every condition was controlled as single variable (Fig.1-5).

2.9. Analysis of the product after scale-up fermentation

Cultured substrate in the optimal medium (1830g) was stored in a polyethylene bag, sealed, and then sterilized at 15psi for 20 min. Bti LLP29 was incubated in the medium at optimal conditions and then crushed to powders.

Bti LLP29 powder was compared with 5100 ITU/mg standards and 1200 ITU/mg wettable powder (WP) that purchased from Wuhan Nature's Favour Bioengineering Co., Ltd (Wuhan, China). The toxicity was determined by bioassay as 2.6 described.

Persistence of Bti LLP29 powder was determined by laboratory bioassay. Two enamel trays were filled with 3000ml of dechlorination water(ȥ��ˮд������). Bti LLP29 powders were added to one experimental tray at 6 mg/L final concentration. Another tray served as control and received only dechlorination water. Each enamel tray was performed in triplicate, to which 30 third stage larvae were added. Mortality was scored 24 hours later. Dead larvae and pupa should be removed every two days, and new larvae in the same number were introduced.

2.10. Statistical analysis

Data on laboratory bioassays were subjected to Analysis of Variance. Analysis was carried out using the software Microsoft Excel 2010. Software SPSS 18.0 for Windows was used for probit analysis to calculating LC50 values.

3. Results and discussion

3.1. Composition of SMS

The constituents of SMS were presented in Table 1. Content of organic material in SMS reached to 960.3mg/g, which was composed of carbohydrate, cellulose, hemicellulose, lignin, some metal ions and so on (Jordan et al., 2008). Qiao et al. (2011) studied that, cellulose, hemicellulose and lignin counted for a lot in SMS. Besides, cellulase was largely produced during the growth of edible mushrooms, which could play a part in bioconversion of lignocellulosic biomass into sugars (Du et al., 2012). As a result, SMS was a good carbon source for Bt cultivation. However, low nitrogen content was detected in SMS, which meant that other nitrogen source needed to be supplemented to Bt cultivation media.

3.2. Four different media combinations for Bt cultivation

Cellulase also could be produced by Bt strains to hydrolyze cellulose in SMS. As Dumas et al. (2009) described, mechanism for this enzymatic hydrolysis was involved in synergistic actions by endoglucanase, exoglucanase and ��-gluosidase. Cellulose chain was cleaved and then glucose was liberated from soluble cellulose, carboxymethyl cellulose and insoluble crystalline cellulose (El-Bendary, 2006). Superadded the sugars existed in SMS before, there was enough carbon source for Bt cultivation. However, as Table 1 shown, low nitrogen content was detected in SMS. For better growth of Bt strains, 4g wheat bran was added in T2 medium to provide organic nitrogen, which was benefit for sporulation and production of toxin in Bt (El-Bendary, 2006).

Inorganic phosphate was considered to be essential for the yield of spores and toxins in Bt production (?zkan et al., 2003). Although the toxicity of Bt was stronger along with the increase of inorganic phosphate concentration (El-Bendary, 2006), considering the cost of production, only 0.2g yeast extract was supplemented in T3 medium. Yeast extract could also be potassium source in that potassium was important for toxin formation by Bt (Wakisaka et al., 1982). Some metal ions also played an important role in sporulation and toxin formulation of Bt. Mg2+, Ca2+ and Mn2+ added in T4 medium in this study, and the effect could be seen in Table 2.

3.3. Growth of Bt after laboratory fermentation

Cell and spore counts were both highest in T4 medium for Bti LLP29, but for strain IPS82, highest cell and spore counts were attained in T3 medium (Table 2). Zhuang et al. (2011) employed wheat bran medium for production of Bt in flask and reported a maximum spore count of 9.2��1010 spores/g substrate. Highest spore counts of 1.25��1011 spores/g substrate was observed in this study, which was greatly higher than the result obtained by 1.8087.

Based on bioassay, toxicities (LC50 values) of Bt were determined. The LC50 values for strains IPS82 and LLP29 were both lowest in T4 medium, which were 2.2mg/L and 2.01mg/L, respectively. According to the results, T4 medium that provided with necessary nutrients was selected to be the best medium for optimizing the fermentation conditions.

3.4. Effect of fermentation conditions on larval mortality

Results of single variable control in fermentation conditions showed that the optimal temperatures for strains LLP29 and IPS82 were found to be 32�� and 30��, respectively (Fig.1). Strains could keep their own cellular pH in neuter and adjust pH of medium automatically in a certain range, which was benefit for their metabolism and biochemical reactions. However, the initial pH value of medium also had indirect influence on strains in some extent. Results showed that the optimum initial pH value was 6 for both Bti strains LLP29 and IPS82 (Fig.2).

In terms of moisture content, the release of spore and crystal would be late and fermentation period would extend when moisture content was too high in medium. However, if it was too low, fermentation would be inhibited since nutrients and water can��t be absorbed by strains (Chen et al., 2009). Results showed that, for strains LLP29 and IPS82, larvicidal activities were both strongest when moisture content was 50% (Fig.3). Particle sizes of medium might also influence the toxicity due to the affected utilization of nutrients for strains. As the results shown, subtle difference could be found for IPS82 in different particle sizes, but for LLP29, particle sizes showed great influence on larvicidal activity (Fig.4). The best result was attained when the ratio of sieved material to initial material was 1: 3. Little influence of inoculum size could be found for Bti LLP29. As for IPS82, the toxicity was significantly higher in the inoculum of 0.75mL-1.25mL than that of 0.25-0.5mL (Fig.5).

3.5. Toxicity and persistence of Bti LLP29 powders by scale-up production

Large scale production of Bti LLP29 was conducted on T4 medium at optimal conditions: temperature 32��, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3 and inoculum size 0.5ml. The comparative toxicities (laboratory bioassays) of Bti LLP29 produced by scale-up fermentation and two commercial products were presented in Table 3. Toxicity of the fermented powders (Bti LLP29 grown substrate) against third fourth instars larvae of C. quinquefasciatus was 1487 ITU/mg, which was higher than the commercial Bti formulation in the market (1200 ITU/mg WP).

Larvicidal persistence of Bti LLP29 powders was determined by laboratory bioassay. High larval mortality was detected after treatment and complete mortality had persisted for 5 days. After 15 days, still 44.83% of mortality was detected, which indicated that Bti LLP29 powders had strong larvicidal persistence. Mass production studies revealed that SMS is a good substrate for Bt cultivation.

4. Conclusions

Bt-based biopesticide was the most successful biopesticide for insect control, and accounted about 2% of the total insecticidal market (Bravo et al., 2011). In this study, cost-effective SMS based media for Bt production by solid-state fermentation were investigated. The media were supplemented with necessary nutrients and the fermentation conditions were optimized. Bti LLP29 powders produced by large scale fermentation showed high ef?cacy of toxicity and larvicidal persistence. Results suggest that SMS is a very cost-effective raw material for mass production of Bt. An additional benefit is that the use of SMS also provides a solution for environmental contamination caused by it.