Environmental Technology
ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: http://www.tandfonline.com/loi/tent20
Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus Zhen Yu, You-chi Zhang, Xiang Zhang & Yin Wang To cite this article: Zhen Yu, You-chi Zhang, Xiang Zhang & Yin Wang (2015): Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus, Environmental Technology, DOI: 10.1080/09593330.2015.1055817 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1055817
Accepted online: 15 Jun 2015.Published online: 24 Jun 2015.
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Date: 08 October 2015, At: 07:55
Environmental Technology, 2015 http://dx.doi.org/10.1080/09593330.2015.1055817
Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus Zhen Yua,b , You-chi Zhanga , Xiang Zhanga,b and Yin Wanga∗ a Key
Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China; b Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
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(Received 24 October 2014; accepted 24 May 2015 ) The feasibility of converting food waste into nematocidal biofertilizer by nematophagous fungus Paecilomyces lilacinus (P. lilacinus) was investigated. The culture conditions of P. lilacinus were optimized through response surface methodology. Results showed that fermentation time, the amount of food waste, initial pH and temperature were most important factors for P. lilacinus production. The P. lilacinus production under optimized conditions was 109.6 ± 0.3 conidia mL−1 . After fermentation, the chemical oxygen demand concentration of food waste was efficiently decreased by 81.92%. Moreover, the property evaluation of the resultant food waste as biofertilizer indicates its high quality with reference to the standard released by the Chinese Ministry of Agriculture. The protease activity and nematocidal ability of P. lilacinus cultured by food waste were 10.8% and 27% higher than those by potato dextrose agar, respectively. Keywords: food waste; Paecilomyces lilacinus; optimization; biofertilizer; biocontrol
1. Introduction Every year, more than one billion tons of food, which is equal to one-third of the annual global food production, is wasted worldwide.[1] Most of the wasted food ends up in landfills, which leads to the formation of putrid landfill gas and leachate that pollute ground and surface waters.[2] Moreover, food waste is a huge loss of potentially valuable resource because food waste contains approximately 60% carbohydrates, 20% proteins, and 10% lipids, which is ideal for many production processes.[3] In recent years, efforts have been made to obtain lactic acid,[4] sugar,[5] ethanol,[6] methane,[7,8] and hydrogen [9] from food waste. However, these studies either obtained products (e.g. enzymes, chemicals, and energy) with massive leftover needed for post treatment, or focused on unitary waste that only comprised a small part of food waste, such as potato peels,[5] orange peel,[10] beet molasses [11] and cabbage waste.[12] The majority of food waste is a mixture of complicated materials and food is produced under excessive consumption of energy, water, and nutrients. Therefore, food waste management requires methods with the ability to dispose the most common kind of food residues and the whole potential of waste organic matter should be exploited more than the use as an energy source. Root knot nematode (RKN, Meloidogyne species) is the most economically important group of plant-parasitic nematodes worldwide that cause huge crop losses by attacking nearly every food and fibre crop.[13] In the
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
past decades, chemical pesticides are the only widely used control against these parasites; however, the growing environmental side effects associated with chemical control have spurred research into biocontrol of plant pathogens. Paecilomyces lilacinus (P. lilacinus) is a soil-inhabiting fungus with promising effects against RKN as a biocontrol agent.[14] However, cost-intensive carbon source makes its cultivation unfavourable and limits its application. Therefore, developing food waste as culture media for P. lilacinus production would facilitate the production of biopesticides in a cost-effective manner. Propagation of P. lilacinus is also a process of food waste mineralization that converts food waste into biofertilizer for agricultural application. Compared with chemical fertilizers, biofertilizers provide socioeconomic and ecological benefits, including improvements of soil and environment quality, food safety, and human and animal health.[15–17] Treatment of food waste with P. lilacinus leaves no residues for second treatment, but valuable and environment-friendly biofertilizer with biocontrol ability against RKN disease. To effectively convert food waste into biofertilizer and facilitate the production of biopesticides, the optimization of P. lilacinus conidia yield from food waste was necessitated by the scarce information on the fermentation of P. lilacinus from mixed substrate. Response surface methodology (RSM) exerts the reduced number of experimental trials needed to evaluate multiple parameters and their interactions, so it is less laborious than other
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approaches to optimize a process.[18] During the optimization process, it is critical to precisely record each response value for later analysis. Thus, an accurate monitoring of P. lilacinus in the food waste is essential. Previous studies to quantify P. lilacinus population densities have used the counting method. It is time consuming and may not have sufficient specificity to accurately quantify the fungus in the food waste.[19] Quantitative real-time polymerase chain reaction (qPCR) relies on the combination of a primer set and an additional dual-labelled fluorogenic probe has been used to successfully determine the abundance of a number of fungal species in complicated environment.[20] In this study, qPCR with P. lilacinus species-specific primers provide a rapid, accurate, and sensitive method of monitoring the yield of P. lilacinus under different conditions. The objectives of this study were to investigate the feasibility of using food waste as the raw material for the production of P. lilacinus biofertilizer for the biocontrol RKN disease. Optimal cultivation conditions for growing P. lilacinus were determined using RSM. The critical factors for fermentation were determined with the Plackett– Burman experimental model, while a central composite design (CCD) was used to optimize the identified critical media components. The yield of P. lilacinus conidia was determined by qPCR. The quality of the fertilizer resulting from P. lilacinus fermentation of food waste was analysed. The morphology and biocontrol efficiency, including eggparasitic ability and protease activity, of P. lilacinus grown on food waste were also assessed. 2.
Sciences and immediately brought to the laboratory for processing. After picking out the bones, the rest of the food waste was crushed using a mechanical mixer, and then sterilized at 121°C for 20 min. Representative characteristics of the collected food waste mixture used in this study are given in Table 1. Protein and nitrogen concentrations were measured by plus the conversion factor 5.7 [3] and these characteristics are consistent with previous reports.[22] 2.3.
QPCR analysis of P. lilacinus PL1210 conidia production
Total DNA was extracted from 0.5 g fermentation samples using a soil DNA extraction kit (Mo-Bio Laboratories Inc., California, USA). qPCR was performed using Roche ® Lightcycler 480. The Stratagene Brilliant QPCR Master Mix TaqMan Kit was used for real-time reactions (Stratagene, Amsterdam, Netherlands). The specific amplification of P. lilacinus was using primers and probe designed by Atkins: PLrtF 5 GAC CCA AAA CTC TTT TTG CAT TAC G 3 ; PLrtR 5 AGA TCC GTT GTT GAA AGT TTT GAT TCATTT GTT TTG 3 ; PLrtP 5 FAM CCG GCG GAATTT CTT CTC TGA GTT GC TAMRA 3 . The PCR amplification and thermal cycle protocol were performed as described by Atkins et al.[23] Reactions were conducted in triplicate. A standard curve was generated by using data derived from the serial dilution of PL1210 so that 1 μL represented 1 × 1010 , 1 × 109 to a final dilution of 100 conidia g−1 sample. The linear correlation coefficient of the standard curve was R2 = 0.987, demonstrating the accuracy of the PCR-based quantification (Figure 1).
Materials and methods
2.1. Microorganism The P. lilacinus strain PL1210 (GenBank ID: KF880384) was isolated from M. incognita egg masses in Xiamen, Fujian, China, and grown on conventional laboratory culture medium potato dextrose agar (PDA) medium. RKN eggs were obtained from infected tomato plants according to the hypochlorite procedure. Briefly, the roots were carefully washed free of soil and then chopped. The RKN eggs were extracted from subsamples by macerating them in a solution containing 5% NaOCl for 10 min.[21] 2.2. Food waste preparation Food waste, mainly containing rice, noodles, meat, and vegetables, was collected from a canteen located at the Institute of Urban Environment, Chinese Academy of Table 1.
Figure 1. Standard curve for the quantitative real-time PCR analysis of P. lilacinus production.
Representative characteristics of collected food waste mixture on a dry basis.
pH 6.02 ± 0.09
TS (%)
Carbohydrates (%)
Proteins (%)
Lipids (%)
13.96 ± 0.11
42.34 ± 0.01
11.77 ± 0.10
15.22 ± 0.04
Note: Number of replicates n = 3; ± standard deviation.
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Environmental Technology The yield of PL1210 production in the following optimization experiment was measured by plotting CT values against the standard curve.
were also measured.[25] GI was calculated as follows:
2.4.
where %G is the number of germinated seeds expressed as % of control values, LT is the average value of root length in the food waste – P. lilacinus mixture, and Lc is the average value of root length in the control.
RSM optimization P. lilacinus production from food waste
Each run of the optimization experiment was performed in 250 mL flask loaded with 100 g fermentation medium and conducted in triplicate. The Plackett–Burman design (PBD) was employed for screening the most significant fermentation parameters affecting P. lilacinus production from food waste. Each independent variable was tested at high and low levels, which was presented as ( + ) and ( − ), respectively. The experimental design with name, symbol code, and actual levels of the variables are given in Table 2. The CCD with the quadratic model was employed to study the combined effect of four significant factors including fermentation days (X 1 ), amount of food waste (X 2 ), initial pH (X 3 ), and temperature (X 5 ) to increase P. lilacinus production. A total of 30 runs are used to optimize the medium. The behaviour of the system was explained by the following second-degree polynomial equation: k k βi Xi + βii Xi2 + βij Xi Xj + ε, Y = β0 + i=1
i=1
i F)
SS
df
MS
F-value
10.04 0.16 0.15 0.016 10.20
14 15 10 5 29
0.72 0.011 0.015 0.003
65.55
< 0.0001
4.52
0.0548
Notes: R2 = 0.9839, R2adj = 0.9689, R2pred = 0.9143, PRESS = 0.87. SS, sum of squares; df, degree of freedom; MS, mean square.
and achieved a COD reduction of 91%. In an attempt to find a more effective treatment for tequila vinasses, Retes et al. [32] observed an 88.7% COD removal by white-rot fungi. High COD level indicates toxic condition and the presence of biologically resistant organic substances. One of the most important environmental problems with food waste lies in its high COD concentration that may pose a negative impact on environment such as polluting surface and underground water. The current study suggests
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(b)
(c)
(d)
(e)
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Figure 2. Three- dimensional response surface plot for P. lilacinus PL1210 yield as a function of variables.
that P. lilacinus could be a promising candidate for COD removal from food waste and lessens the major environmental problem brought by high COD loads from food waste. As shown in Figure 3(b), the pH dropped from 6.04 at the onset of fermentation to about 5.24 at day 8, increased to 6.63 at day 16, and then decreased to 5.72 at the end of fermentation. The increase in pH was caused by the biodegradation of acids such as those with phenolic and carboxylic groups, as well as the mineralization of organic compounds (e.g. amino acids and peptides) to inorganic compounds.[33] The decrease in pH may be due to the
decomposition of starch, followed by acidic fermentation of sugars and volatilization of ammoniacal nitrogen accompanied with H+ released from microbial nitrification and carbon dioxide release during the fermentation process.[24] Furthermore, the large quantities of carbon dioxide that are given off during the composting process and production of various organic and inorganic acids by P. lilacinus also contributed to the decrease in pH.[34] The EC indicates the mineralization rate and possible phytotoxic/phyto-inhibitory effects as low germination rate and withering on the growth of plant as a fertilizer.[35] The EC of fermentation increased initially from 1309 to
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(b)
(c)
(d)
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Figure 3. Changes in COD concentration (a), pH (b), EC (c), and TOC (d) of food waste after inoculation of P. lilacinus PL1210. Data points are mean values ± SD.
1579 μs cm−1 at day 13, followed by a gradual decrease to 1473 μs cm−1 until the end of the fermentation process (Figure 3(c)). The initial EC increase may be attributed to the release of mineral salts through the decomposition of organic matters. As fermentation progressed, volatilization of ammonia and precipitation of mineral salts could lead to the decrease in EC at a later phase of composting. In this study, the EC of the fermentation product did not exceed the limit content of 3000 μs cm−1 at day 18, which indicates that EC would not adversely affect plant growth. Table 5 shows that the contents of carbon, nitrogen, and ash increased by 10.16%, 92.72%, and 103.21%, respectively. However, a decrease in C/N ratio (by 42.77%) Table 5. Physico-chemical parameters of the food waste mixture on a dry basis. Parameters
At start
C (%) 42.34 ± 0.01 N (%) 2.06 ± 0.01 C/N 20.55 TS (%) 13.96 ± 0.11 Ash (%) 2.80 ± 0.02 S (%) 0.205 ± 0.00 P2 O5 (%) – K2 O (%) – Organic matter (%) GI (%) –
At end
%
46.64 ± 0.03* 3.97 ± 0.01* 11.76* 9.63 ± 0.40* 5.69 ± 0.74* 0.32 ± 0.02* 0.37 ± 0.05 1.1 ± 0.13 58.44 ± 6.55
10.16( + ) 92.72( + ) 42.77( − ) 31.02( − ) 103.21( + ) 56.10( + ) – –
145.96
–
Number of replicates, n = 3; Values are shown as mean ± SD; ‘*’ Indicates significant differences among the values of same parameters (P < 0.05).
and TS (31.02%) was observed compared with the original food waste. Increase in ash content together with large TOC losses (Figure 3(d)) suggested the occurrence of intensive humification the during the composting process.[36] Carbon is a source of energy for microorganisms to develop and propagate. Carbon was almost entirely absorbed by the microorganisms and transformed to CO2 during the metabolism process. The leftover carbon will be converted into membrane and protoplasm form. Thus, while the organic matter in food waste is decomposed by microorganisms through which organic carbon is oxidized in the aerobic condition to CO2 gas, nitrogen should be transformed and assimilated by the growing fungal cells, which caused the decrease in C/N ratio in this experiment. Rapid and entire humification of a substrate essentially depends on its initial C/N ratio. Initial C/N ratio is a key factor for fungal proper growth and degradable activities, as well as in controlling ammonia loss within the composting process.[37] In the current study, the initial C/N ratio was 20.55, which was a suitable value for composting.[38] After 18 d of composting, the C/N ratio dropped to 11.76 at the end of the biological process. C/N ratio is a pivotal indicator of compost maturity and quality. According to maturity indices established for compost, the C/N ratio should reach a value below 20 and preferably below 10.[39] The value of C/N in this study (11.76) was satisfactory because it met the standard. The produced biofertilizer was analysed for agronomically important parameters. N, P, and K are three critical nutrient elements required for seed production, root development, and plant growth. The total nutrients of N, P2 O5 , and K2 O was more than 5%, and the organic
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matter content was up to 58% (Table 5), which completely reached the technical quality index of NY5252012 Organic Fertilizer standard released by the Chinese Ministry of Agriculture.[40] GI value is an important biological indicator and regarded as the most sensitive parameter used to evaluate the maturity and toxicity of compost. A GI value of 50% was previously used as an indicator of phytotoxin-free compost. When the GI value is higher than 80%, the compost seemed to be completely non-toxic to plants.[25] In the present study, a GI value of 145.96% was observed using the food waste biofertilizer as the substrate for corn seeds (Table 5). Such an increased GI value of the biofertilizer could be ascribed to plant growthpromoting substances such as indole acetic acid produced
by P. lilacinus as confirmed by previous studies and our lab experiment.[41] Therefore, the product by P. lilacinus from food waste is an excellent fertilizer for plant growth. 3.3. Morphological observation by SEM Food waste is a mixture of complicated materials. Thus, food waste may contain microbial growth inhibitory properties, such as high salinity and toxic substances, which may pose adverse effects on the development and growth of P. lilacinus, leading to a malformation of P. lilacinus morphology. In the present study, no significant difference in the morphological structure between PL1210 grown on food waste and PDA was observed through SEM (Figure 4). As grown on PDA (Figure 4(a)), PL1210
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4. SEM of PL1210 conidiophores (a), mycelium (b) and conidia (c) from PDA medium; conidiophores (d), mycelium (e) and conidia (f) from food waste medium.
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cultured on food waste showed the following morphological characteristics (Figure 4(b)): lageniform phialides are tapering to a neck producing lemon-shaped conidia and stretched, regular, and homogeneous hyphae of constant diameter with smooth external surface. 3.4. Protease activity analysis and egg-parasitic ability Insect cuticle is the main barrier for fungal invasion and mainly consists of protein and chitin. The structure of the nematode eggshell is similar to that of insect cuticle. Morgan et al. first reported that the eggshell of M. arenaria exhibited a number of ultrastructural changes after being infected by P. lilacinus.[42] At present, P. lilacinus is believed to produce proteases and chitinases to degrade the nematode eggshell. Moreover, a basic serine protease pSP-3 has been confirmed to play a vital role during the infection process.[43] Therefore, protease is important for P. lilacinus to exert its biocontrol function and examine the feasibility in using food waste as the nutrient source for P. lilacinus production in this study. The quantitative analysis of protease activity using the Folin–phenol reagent showed that the maximum protease activity of PL1210 cultured by PDA was at day 7 and that by food waste was at day 8. In addition, the maximum value of protease activity in the food waste medium was 10.8% higher than that in the PDA medium (Figure 5). The overexpression of insect cuticle-degrading enzymes in insecticidal fungi may significantly enhance the virulence against insects.[44] Similarly, an increase in nematode eggshell-degrading enzymes could also enhance the virulence of P. lilacinus against plant-parasitic nematode eggs, as confirmed by a previous research.[45] Table 6 shows the comparison of the egg-parasitic abilities of PL1210 cultured by food waste and PDA. The relative parasitizing rate of PL1210 cultured by food waste was increased by about 27% as compared with that by PDA (P < 0.05). The observed increase in the relative parasitizing rate may be due to the higher concentration
Table 6. Bioassay results of egg-parasitic abilities.
Number of second-stage juveniles Relative parasitizing rate (%)
CK
P. lilacinus cultured by PDA
P. lilacinus cultured by food waste
356 ± 12a
183 ± 19b
137 ± 14c
48.60 ± 5.34a 61.52 ± 3.93b
Note: Number of replicates n = 3; Values are shown as mean ± SD. The treatment means were separated according to Duncan’s new multiple range test. Means in a row with different letter superscripts are significantly different at P < 0.05.
of cuticle-degrading proteases expressed in PL1210–food waste mixture. 4.
Conclusion
In conclusion, this research investigated the feasibility of using biocontrol fungus P. lilacinus to convert food waste into nematocidal biofertilizer. The RSM was used for the systematic optimization of the culture conditions of P. lilacinus production from food waste. With effective COD removal and proper maturation, the food waste remaining after P. lilacinus fermentation showed promising quality as biofertilizer with high N, P, and K nutrient content. In addition, the protease activity assay and eggparasitic ability test showed that the biopesticide produced from food waste under the optimal condition was better than that produced by the conventional medium PDA. These results indicated that the use of biocontrol fungus P. lilacinus to convert food waste into biofertilizer with nematocidal activity is an efficient way to facilitated food waste reutilization. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported by the 863 – National High Technology Research and Development Programme of China [No. 2012AA06A204-2] and Xiamen Science and Technology Major Programme [No. 3502Z20131018] and Main Project of Chinese Academy Sciences [KZZD-EW-16].
References
Figure 5. Activity of crude protease extracts of PL1210 from conventional and food waste media at various time points. Data points are mean values ± SD.
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[42] Morganjones G, White JF, Rodriguezkabana R. Phytonematode pathology – ultrastructural studies .2. Parasitism of Meloidogyne-Arenaria eggs and Larvae by PaecilomycesLilacinus. Nematropica. 1984;14:57–71. [43] Dong LQ, Yang JK, Zhang KQ. Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus. J Appl Microbiol. 2007;103:2476– 2488. [44] Shang Y, Duan Z, Huang W, Gao Q, Wang C. Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous Tyrosinase gene. J Invertebr Pathol. 2012;109:105–109. [45] Yang J, Zhao X, Liang L, et al. Overexpression of a cuticledegrading protease Ver112 increases the nematicidal activity of Paecilomyces lilacinus. Appl Microbiol Biotechnol. 2011;89:1895–1903.
ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: http://www.tandfonline.com/loi/tent20
Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus Zhen Yu, You-chi Zhang, Xiang Zhang & Yin Wang To cite this article: Zhen Yu, You-chi Zhang, Xiang Zhang & Yin Wang (2015): Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus, Environmental Technology, DOI: 10.1080/09593330.2015.1055817 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1055817
Accepted online: 15 Jun 2015.Published online: 24 Jun 2015.
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Date: 08 October 2015, At: 07:55
Environmental Technology, 2015 http://dx.doi.org/10.1080/09593330.2015.1055817
Conversion of food waste into biofertilizer for the biocontrol of root knot nematode by Paecilomyces lilacinus Zhen Yua,b , You-chi Zhanga , Xiang Zhanga,b and Yin Wanga∗ a Key
Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China; b Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
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(Received 24 October 2014; accepted 24 May 2015 ) The feasibility of converting food waste into nematocidal biofertilizer by nematophagous fungus Paecilomyces lilacinus (P. lilacinus) was investigated. The culture conditions of P. lilacinus were optimized through response surface methodology. Results showed that fermentation time, the amount of food waste, initial pH and temperature were most important factors for P. lilacinus production. The P. lilacinus production under optimized conditions was 109.6 ± 0.3 conidia mL−1 . After fermentation, the chemical oxygen demand concentration of food waste was efficiently decreased by 81.92%. Moreover, the property evaluation of the resultant food waste as biofertilizer indicates its high quality with reference to the standard released by the Chinese Ministry of Agriculture. The protease activity and nematocidal ability of P. lilacinus cultured by food waste were 10.8% and 27% higher than those by potato dextrose agar, respectively. Keywords: food waste; Paecilomyces lilacinus; optimization; biofertilizer; biocontrol
1. Introduction Every year, more than one billion tons of food, which is equal to one-third of the annual global food production, is wasted worldwide.[1] Most of the wasted food ends up in landfills, which leads to the formation of putrid landfill gas and leachate that pollute ground and surface waters.[2] Moreover, food waste is a huge loss of potentially valuable resource because food waste contains approximately 60% carbohydrates, 20% proteins, and 10% lipids, which is ideal for many production processes.[3] In recent years, efforts have been made to obtain lactic acid,[4] sugar,[5] ethanol,[6] methane,[7,8] and hydrogen [9] from food waste. However, these studies either obtained products (e.g. enzymes, chemicals, and energy) with massive leftover needed for post treatment, or focused on unitary waste that only comprised a small part of food waste, such as potato peels,[5] orange peel,[10] beet molasses [11] and cabbage waste.[12] The majority of food waste is a mixture of complicated materials and food is produced under excessive consumption of energy, water, and nutrients. Therefore, food waste management requires methods with the ability to dispose the most common kind of food residues and the whole potential of waste organic matter should be exploited more than the use as an energy source. Root knot nematode (RKN, Meloidogyne species) is the most economically important group of plant-parasitic nematodes worldwide that cause huge crop losses by attacking nearly every food and fibre crop.[13] In the
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
past decades, chemical pesticides are the only widely used control against these parasites; however, the growing environmental side effects associated with chemical control have spurred research into biocontrol of plant pathogens. Paecilomyces lilacinus (P. lilacinus) is a soil-inhabiting fungus with promising effects against RKN as a biocontrol agent.[14] However, cost-intensive carbon source makes its cultivation unfavourable and limits its application. Therefore, developing food waste as culture media for P. lilacinus production would facilitate the production of biopesticides in a cost-effective manner. Propagation of P. lilacinus is also a process of food waste mineralization that converts food waste into biofertilizer for agricultural application. Compared with chemical fertilizers, biofertilizers provide socioeconomic and ecological benefits, including improvements of soil and environment quality, food safety, and human and animal health.[15–17] Treatment of food waste with P. lilacinus leaves no residues for second treatment, but valuable and environment-friendly biofertilizer with biocontrol ability against RKN disease. To effectively convert food waste into biofertilizer and facilitate the production of biopesticides, the optimization of P. lilacinus conidia yield from food waste was necessitated by the scarce information on the fermentation of P. lilacinus from mixed substrate. Response surface methodology (RSM) exerts the reduced number of experimental trials needed to evaluate multiple parameters and their interactions, so it is less laborious than other
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approaches to optimize a process.[18] During the optimization process, it is critical to precisely record each response value for later analysis. Thus, an accurate monitoring of P. lilacinus in the food waste is essential. Previous studies to quantify P. lilacinus population densities have used the counting method. It is time consuming and may not have sufficient specificity to accurately quantify the fungus in the food waste.[19] Quantitative real-time polymerase chain reaction (qPCR) relies on the combination of a primer set and an additional dual-labelled fluorogenic probe has been used to successfully determine the abundance of a number of fungal species in complicated environment.[20] In this study, qPCR with P. lilacinus species-specific primers provide a rapid, accurate, and sensitive method of monitoring the yield of P. lilacinus under different conditions. The objectives of this study were to investigate the feasibility of using food waste as the raw material for the production of P. lilacinus biofertilizer for the biocontrol RKN disease. Optimal cultivation conditions for growing P. lilacinus were determined using RSM. The critical factors for fermentation were determined with the Plackett– Burman experimental model, while a central composite design (CCD) was used to optimize the identified critical media components. The yield of P. lilacinus conidia was determined by qPCR. The quality of the fertilizer resulting from P. lilacinus fermentation of food waste was analysed. The morphology and biocontrol efficiency, including eggparasitic ability and protease activity, of P. lilacinus grown on food waste were also assessed. 2.
Sciences and immediately brought to the laboratory for processing. After picking out the bones, the rest of the food waste was crushed using a mechanical mixer, and then sterilized at 121°C for 20 min. Representative characteristics of the collected food waste mixture used in this study are given in Table 1. Protein and nitrogen concentrations were measured by plus the conversion factor 5.7 [3] and these characteristics are consistent with previous reports.[22] 2.3.
QPCR analysis of P. lilacinus PL1210 conidia production
Total DNA was extracted from 0.5 g fermentation samples using a soil DNA extraction kit (Mo-Bio Laboratories Inc., California, USA). qPCR was performed using Roche ® Lightcycler 480. The Stratagene Brilliant QPCR Master Mix TaqMan Kit was used for real-time reactions (Stratagene, Amsterdam, Netherlands). The specific amplification of P. lilacinus was using primers and probe designed by Atkins: PLrtF 5 GAC CCA AAA CTC TTT TTG CAT TAC G 3 ; PLrtR 5 AGA TCC GTT GTT GAA AGT TTT GAT TCATTT GTT TTG 3 ; PLrtP 5 FAM CCG GCG GAATTT CTT CTC TGA GTT GC TAMRA 3 . The PCR amplification and thermal cycle protocol were performed as described by Atkins et al.[23] Reactions were conducted in triplicate. A standard curve was generated by using data derived from the serial dilution of PL1210 so that 1 μL represented 1 × 1010 , 1 × 109 to a final dilution of 100 conidia g−1 sample. The linear correlation coefficient of the standard curve was R2 = 0.987, demonstrating the accuracy of the PCR-based quantification (Figure 1).
Materials and methods
2.1. Microorganism The P. lilacinus strain PL1210 (GenBank ID: KF880384) was isolated from M. incognita egg masses in Xiamen, Fujian, China, and grown on conventional laboratory culture medium potato dextrose agar (PDA) medium. RKN eggs were obtained from infected tomato plants according to the hypochlorite procedure. Briefly, the roots were carefully washed free of soil and then chopped. The RKN eggs were extracted from subsamples by macerating them in a solution containing 5% NaOCl for 10 min.[21] 2.2. Food waste preparation Food waste, mainly containing rice, noodles, meat, and vegetables, was collected from a canteen located at the Institute of Urban Environment, Chinese Academy of Table 1.
Figure 1. Standard curve for the quantitative real-time PCR analysis of P. lilacinus production.
Representative characteristics of collected food waste mixture on a dry basis.
pH 6.02 ± 0.09
TS (%)
Carbohydrates (%)
Proteins (%)
Lipids (%)
13.96 ± 0.11
42.34 ± 0.01
11.77 ± 0.10
15.22 ± 0.04
Note: Number of replicates n = 3; ± standard deviation.
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Environmental Technology The yield of PL1210 production in the following optimization experiment was measured by plotting CT values against the standard curve.
were also measured.[25] GI was calculated as follows:
2.4.
where %G is the number of germinated seeds expressed as % of control values, LT is the average value of root length in the food waste – P. lilacinus mixture, and Lc is the average value of root length in the control.
RSM optimization P. lilacinus production from food waste
Each run of the optimization experiment was performed in 250 mL flask loaded with 100 g fermentation medium and conducted in triplicate. The Plackett–Burman design (PBD) was employed for screening the most significant fermentation parameters affecting P. lilacinus production from food waste. Each independent variable was tested at high and low levels, which was presented as ( + ) and ( − ), respectively. The experimental design with name, symbol code, and actual levels of the variables are given in Table 2. The CCD with the quadratic model was employed to study the combined effect of four significant factors including fermentation days (X 1 ), amount of food waste (X 2 ), initial pH (X 3 ), and temperature (X 5 ) to increase P. lilacinus production. A total of 30 runs are used to optimize the medium. The behaviour of the system was explained by the following second-degree polynomial equation: k k βi Xi + βii Xi2 + βij Xi Xj + ε, Y = β0 + i=1
i=1
i F)
SS
df
MS
F-value
10.04 0.16 0.15 0.016 10.20
14 15 10 5 29
0.72 0.011 0.015 0.003
65.55
< 0.0001
4.52
0.0548
Notes: R2 = 0.9839, R2adj = 0.9689, R2pred = 0.9143, PRESS = 0.87. SS, sum of squares; df, degree of freedom; MS, mean square.
and achieved a COD reduction of 91%. In an attempt to find a more effective treatment for tequila vinasses, Retes et al. [32] observed an 88.7% COD removal by white-rot fungi. High COD level indicates toxic condition and the presence of biologically resistant organic substances. One of the most important environmental problems with food waste lies in its high COD concentration that may pose a negative impact on environment such as polluting surface and underground water. The current study suggests
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(b)
(c)
(d)
(e)
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Figure 2. Three- dimensional response surface plot for P. lilacinus PL1210 yield as a function of variables.
that P. lilacinus could be a promising candidate for COD removal from food waste and lessens the major environmental problem brought by high COD loads from food waste. As shown in Figure 3(b), the pH dropped from 6.04 at the onset of fermentation to about 5.24 at day 8, increased to 6.63 at day 16, and then decreased to 5.72 at the end of fermentation. The increase in pH was caused by the biodegradation of acids such as those with phenolic and carboxylic groups, as well as the mineralization of organic compounds (e.g. amino acids and peptides) to inorganic compounds.[33] The decrease in pH may be due to the
decomposition of starch, followed by acidic fermentation of sugars and volatilization of ammoniacal nitrogen accompanied with H+ released from microbial nitrification and carbon dioxide release during the fermentation process.[24] Furthermore, the large quantities of carbon dioxide that are given off during the composting process and production of various organic and inorganic acids by P. lilacinus also contributed to the decrease in pH.[34] The EC indicates the mineralization rate and possible phytotoxic/phyto-inhibitory effects as low germination rate and withering on the growth of plant as a fertilizer.[35] The EC of fermentation increased initially from 1309 to
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(b)
(c)
(d)
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Figure 3. Changes in COD concentration (a), pH (b), EC (c), and TOC (d) of food waste after inoculation of P. lilacinus PL1210. Data points are mean values ± SD.
1579 μs cm−1 at day 13, followed by a gradual decrease to 1473 μs cm−1 until the end of the fermentation process (Figure 3(c)). The initial EC increase may be attributed to the release of mineral salts through the decomposition of organic matters. As fermentation progressed, volatilization of ammonia and precipitation of mineral salts could lead to the decrease in EC at a later phase of composting. In this study, the EC of the fermentation product did not exceed the limit content of 3000 μs cm−1 at day 18, which indicates that EC would not adversely affect plant growth. Table 5 shows that the contents of carbon, nitrogen, and ash increased by 10.16%, 92.72%, and 103.21%, respectively. However, a decrease in C/N ratio (by 42.77%) Table 5. Physico-chemical parameters of the food waste mixture on a dry basis. Parameters
At start
C (%) 42.34 ± 0.01 N (%) 2.06 ± 0.01 C/N 20.55 TS (%) 13.96 ± 0.11 Ash (%) 2.80 ± 0.02 S (%) 0.205 ± 0.00 P2 O5 (%) – K2 O (%) – Organic matter (%) GI (%) –
At end
%
46.64 ± 0.03* 3.97 ± 0.01* 11.76* 9.63 ± 0.40* 5.69 ± 0.74* 0.32 ± 0.02* 0.37 ± 0.05 1.1 ± 0.13 58.44 ± 6.55
10.16( + ) 92.72( + ) 42.77( − ) 31.02( − ) 103.21( + ) 56.10( + ) – –
145.96
–
Number of replicates, n = 3; Values are shown as mean ± SD; ‘*’ Indicates significant differences among the values of same parameters (P < 0.05).
and TS (31.02%) was observed compared with the original food waste. Increase in ash content together with large TOC losses (Figure 3(d)) suggested the occurrence of intensive humification the during the composting process.[36] Carbon is a source of energy for microorganisms to develop and propagate. Carbon was almost entirely absorbed by the microorganisms and transformed to CO2 during the metabolism process. The leftover carbon will be converted into membrane and protoplasm form. Thus, while the organic matter in food waste is decomposed by microorganisms through which organic carbon is oxidized in the aerobic condition to CO2 gas, nitrogen should be transformed and assimilated by the growing fungal cells, which caused the decrease in C/N ratio in this experiment. Rapid and entire humification of a substrate essentially depends on its initial C/N ratio. Initial C/N ratio is a key factor for fungal proper growth and degradable activities, as well as in controlling ammonia loss within the composting process.[37] In the current study, the initial C/N ratio was 20.55, which was a suitable value for composting.[38] After 18 d of composting, the C/N ratio dropped to 11.76 at the end of the biological process. C/N ratio is a pivotal indicator of compost maturity and quality. According to maturity indices established for compost, the C/N ratio should reach a value below 20 and preferably below 10.[39] The value of C/N in this study (11.76) was satisfactory because it met the standard. The produced biofertilizer was analysed for agronomically important parameters. N, P, and K are three critical nutrient elements required for seed production, root development, and plant growth. The total nutrients of N, P2 O5 , and K2 O was more than 5%, and the organic
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matter content was up to 58% (Table 5), which completely reached the technical quality index of NY5252012 Organic Fertilizer standard released by the Chinese Ministry of Agriculture.[40] GI value is an important biological indicator and regarded as the most sensitive parameter used to evaluate the maturity and toxicity of compost. A GI value of 50% was previously used as an indicator of phytotoxin-free compost. When the GI value is higher than 80%, the compost seemed to be completely non-toxic to plants.[25] In the present study, a GI value of 145.96% was observed using the food waste biofertilizer as the substrate for corn seeds (Table 5). Such an increased GI value of the biofertilizer could be ascribed to plant growthpromoting substances such as indole acetic acid produced
by P. lilacinus as confirmed by previous studies and our lab experiment.[41] Therefore, the product by P. lilacinus from food waste is an excellent fertilizer for plant growth. 3.3. Morphological observation by SEM Food waste is a mixture of complicated materials. Thus, food waste may contain microbial growth inhibitory properties, such as high salinity and toxic substances, which may pose adverse effects on the development and growth of P. lilacinus, leading to a malformation of P. lilacinus morphology. In the present study, no significant difference in the morphological structure between PL1210 grown on food waste and PDA was observed through SEM (Figure 4). As grown on PDA (Figure 4(a)), PL1210
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4. SEM of PL1210 conidiophores (a), mycelium (b) and conidia (c) from PDA medium; conidiophores (d), mycelium (e) and conidia (f) from food waste medium.
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cultured on food waste showed the following morphological characteristics (Figure 4(b)): lageniform phialides are tapering to a neck producing lemon-shaped conidia and stretched, regular, and homogeneous hyphae of constant diameter with smooth external surface. 3.4. Protease activity analysis and egg-parasitic ability Insect cuticle is the main barrier for fungal invasion and mainly consists of protein and chitin. The structure of the nematode eggshell is similar to that of insect cuticle. Morgan et al. first reported that the eggshell of M. arenaria exhibited a number of ultrastructural changes after being infected by P. lilacinus.[42] At present, P. lilacinus is believed to produce proteases and chitinases to degrade the nematode eggshell. Moreover, a basic serine protease pSP-3 has been confirmed to play a vital role during the infection process.[43] Therefore, protease is important for P. lilacinus to exert its biocontrol function and examine the feasibility in using food waste as the nutrient source for P. lilacinus production in this study. The quantitative analysis of protease activity using the Folin–phenol reagent showed that the maximum protease activity of PL1210 cultured by PDA was at day 7 and that by food waste was at day 8. In addition, the maximum value of protease activity in the food waste medium was 10.8% higher than that in the PDA medium (Figure 5). The overexpression of insect cuticle-degrading enzymes in insecticidal fungi may significantly enhance the virulence against insects.[44] Similarly, an increase in nematode eggshell-degrading enzymes could also enhance the virulence of P. lilacinus against plant-parasitic nematode eggs, as confirmed by a previous research.[45] Table 6 shows the comparison of the egg-parasitic abilities of PL1210 cultured by food waste and PDA. The relative parasitizing rate of PL1210 cultured by food waste was increased by about 27% as compared with that by PDA (P < 0.05). The observed increase in the relative parasitizing rate may be due to the higher concentration
Table 6. Bioassay results of egg-parasitic abilities.
Number of second-stage juveniles Relative parasitizing rate (%)
CK
P. lilacinus cultured by PDA
P. lilacinus cultured by food waste
356 ± 12a
183 ± 19b
137 ± 14c
48.60 ± 5.34a 61.52 ± 3.93b
Note: Number of replicates n = 3; Values are shown as mean ± SD. The treatment means were separated according to Duncan’s new multiple range test. Means in a row with different letter superscripts are significantly different at P < 0.05.
of cuticle-degrading proteases expressed in PL1210–food waste mixture. 4.
Conclusion
In conclusion, this research investigated the feasibility of using biocontrol fungus P. lilacinus to convert food waste into nematocidal biofertilizer. The RSM was used for the systematic optimization of the culture conditions of P. lilacinus production from food waste. With effective COD removal and proper maturation, the food waste remaining after P. lilacinus fermentation showed promising quality as biofertilizer with high N, P, and K nutrient content. In addition, the protease activity assay and eggparasitic ability test showed that the biopesticide produced from food waste under the optimal condition was better than that produced by the conventional medium PDA. These results indicated that the use of biocontrol fungus P. lilacinus to convert food waste into biofertilizer with nematocidal activity is an efficient way to facilitated food waste reutilization. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported by the 863 – National High Technology Research and Development Programme of China [No. 2012AA06A204-2] and Xiamen Science and Technology Major Programme [No. 3502Z20131018] and Main Project of Chinese Academy Sciences [KZZD-EW-16].
References
Figure 5. Activity of crude protease extracts of PL1210 from conventional and food waste media at various time points. Data points are mean values ± SD.
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