Preparation and utilization of phosphate biofertilizers using agricultural waste

Journal of Integrative Agriculture 2015, 14(1): 158–167 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Preparation and utilization of phosphate biofertilizers using agricultural waste WANG Hong-yuan, LIU Shen, ZHAI Li-mei, ZHANG Ji-zong, REN Tian-zhi, FAN Bing-quan, LIU Hong-bin Key Laboratory of Nonpoint Source Pollution Control, Ministry of Agriculture/Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

Abstract In this study, Aspergillus niger 1107 was isolated and identified as an efficient phosphate-solubilizing fungus (PSF). This strain generated 689 mg soluble P L–1 NBRIP medium after 10 d of culture. To produce an affordable biofertilizer using A. niger 1107, the potential of widely available carrier materials for growth and maintenance of this strain were evaluated. The effects of sterilization procedures (autoclaving and gamma-ray irradiation) on the suitability of these carriers to maintain growth of the fungus were also investigated. The carrier materials were peat, corn cobs with 20% (w/w) perlite (CCP), wheat husks with 20% (w/w) perlite (WHP), and composted cattle manure with 20% (w/w) perlite (CCMP). In the first 5-6 mon of storage, the carriers sterilized by gamma-ray irradiation maintained higher inoculum loads than those in carriers sterilized by autoclaving. However, this effect was not detectable after 7 mon of storage. For the P-biofertilizer on WHP, more than 2.0×107 viable spores of A. niger g–1 inoculant survived after 7 mon of storage. When this biofertilizer was applied to Chinese cabbage in a pot experiment, there were 5.6×106 spores of A. niger g–1 soil before plant harvesting. In the pot experiment, Chinese cabbage plants grown in soil treated with peat- and WHP-based P-biofertilizers showed significantly greater growth (P<0.05) than that of plants grown in soil treated with free-cell biofertilizer or the CCMP-based biofertilizer. Also, the peat- and WHP-based P-biofertilizers increased the available P content in soil. Keywords: biofertilizer carrier, sterilization method, phosphate biofertilizer, P-solubilizing fungi, Aspergillus niger

1. Introduction After nitrogen, phosphorus (P) is the second most important macronutrient for plant growth. Many soils are P-deficient

Received 17 September, 2013 Accepted 12 January, 2014 WANG Hong-yuan, Tel: +86-10-82106737, E-mail: wanghong [email protected]; Correspondence LIU Hong-bin, Tel: +86-1082108763, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60760-7

because low levels of soluble phosphate are available for plant growth, although they may have high levels of total P. It is reported that many soils have high reserves of total P, accounting for approximately 0.05% of the soil dry weight on average. However, only a small proportion (1-5%) of total soil P is available in a soluble form that can be taken up by plants (Takahashi and Anwar 2007; Mahidi et al. 2011). Soil P deficiency has traditionally been addressed by adding P-fertilizers. However, only a fraction of the added P is eventually assimilated by plants, and the rest (almost 75-90%) is precipitated by Fe, Al, and Ca complexes in the soils (Gyaneshwar et al. 2002). The low P-use efficiency of plants cultivated on farmland has not only resulted in greater application of P fertilizers, but has also led to a variety

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of environmental problems, such as water eutrophication (Chang and Yang 2009; Kang et al. 2011). Enhancement of plant growth through fertilization to meet the increasing demands for food has resulted in intense mining of P-containing minerals around the world. It has been estimated that these P mines could be depleted by 2060 (Gilvert 2009). Many agricultural soils represent a source of P that is not readily available to plants but may still be recovered. It has been suggested that the amount of P in agricultural soils is sufficient to sustain maximum crop yields worldwide for about 100 years (Walpola and Yoon 2012). To use the P accumulated in soils, P-solubilizing microorganisms (PSMs) that are able to transform insoluble P to soluble forms can function as biofertilizers to increase the soluble P content (Narsian and Patel 2000; Delvasto 2006; Khan et al. 2007; Zhu et al. 2012). The use of P-biofertilizers is a promising approach to improve world food security through enhancing agricultural yield in developing countries in Africa and Asia, which together account for 50 and 74%, respectively, of the global land mass and population (Ogbo 2010). Many studies have shown that growth and P uptake by plants can be enhanced by inoculation of phosphate-solubilizing fungi (PSF). This effect has been shown in pot experiments (Vassilev et al. 2006; Mittal et al. 2008) and under field conditions (Duponnois et al. 2005; Valverde et al. 2006). Among these fungi, P-solubilizing Aspergillus species have been widely studied because of their strong ability to provide available P and improve plant growth (EI-Azouni 2008; Mittal et al. 2008; Ogbo 2010; Jain et al. 2012; Xiao et al. 2013). El-Azouni et al. (2008) reported that Aspergillus niger was able to solubilize and release inorganic P; the studied strain released 490 μg P mL–1 after 7 d of growth in Pikovskaya (PVK) medium supplemented with tricalcium phosphate (TCP). In a pot experiment, inoculation with A. niger significantly increased plant height by up to 27.5% and plant dry weight by up to 22.7%, compared with plants grown in non-inoculated TCP soil. Mittal et al. (2008) isolated six P-solubilizing fungi, two strains of A. awamori and four strains of Penicillium citrinum, from the rhizosphere of various crops. When these strains were inoculated onto chickpea plants in a pot experiment, the two A. awamori strains had the strongest growth-promoting effects. The plants inoculated with A. awamori showed a 7–12% increase in shoot height, a nearly three-fold increase in seed number, and a two-fold increase in seed weight, as compared with uninoculated control plants. Ogbo (2010) and Xiao et al. (2013) reported that biofertilizer produced by A. niger significantly (P<0.05) improved the growth of pigeon pea and wheat plants under culture conditions. Jain et al. (2012) demonstrated that A. awamori S29 significantly increased mungbean growth, total P content, and plant biomass in a pot experiment.

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Biofertilizers are usually prepared as carrier-based inoculants containing effective microorganisms (Accinelli et al. 2009). However, the local development of commercial biofertilizers is often restricted by technological limitations or the scarcity of local sources of peat, the most commonly used biofertilizer carrier in many countries (Khavazi et al. 2007). The development of locally produced inoculants is desirable, as they are adapted to local conditions. To do so, it is important to use carriers and preparation methods that are widely available and accessible locally. A suitable biofertilizer carrier should meet the following criteria: (1) it should be available in powder or granule forms; (2) it should be able to support microorganism growth and survival, and easily release functional microorganisms into the soil; (3) it should have a strong moisture absorption capability, good aeration characteristics, and excellent pH buffering capacity; (4) it should be non-toxic and environmentally friendly; (5) it should be easily sterilized, manufactured, and handled in the field, and have good storage qualities; and (6) it should be inexpensive (Stephens and Rask 2000; Rebah et al. 2002; Rivera-Cruz et al. 2008). Organic wastes from animal production and agriculture and byproducts of agricultural and food processing industries cause substantial environmental and social problems in both developed and developing countries. However, most of these organic wastes meet the requirements of a biofertilizer carrier. Therefore, they could be good carrier materials. Perlite is an organic stone of volcanic origin; it is made of aluminum silicate and contains little water. In a previous study, this material was a good carrier material because of its light weight, its porosity, and its environmental-friendliness (Khavazi et al. 2007). Therefore, in this study, we evaluated the performance of perlite as a major component of carrier materials (corn cobs, wheat husks, and composted cattle manure). Carrier materials for biofertilizers must meet the criteria described above, but they also must be sterilized to retain a large population of the inoculant microorganism during long-term storage. Gamma-ray irradiation appears to be a promising sterilization technology because it is easy, it can be used on a large scale, and it has been shown to result in reduced storage losses, extended shelf-life, and/ or larger populations of the inoculant during storage (Khavazi et al. 2007; Fernandes et al. 2011). This technology has been used to sterilize commodities such as tubers and bulbs, grains, dry ingredients, meat, and fruit (Farkas 2006). However, gamma-ray irradiation has rarely been used to sterilize carrier materials for biofertilizers, and its effects on the attributes of carriers are unknown (Khavazi et al. 2007). Thus, we evaluated the effects of gamma-ray irradiation and autoclaving on the ability of the carrier materials to sustain populations of the inoculant during storage, and on the ability

WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167

2. Results 2.1. Isolation of P-solubilizing fungi Based on morphological features, 30 morphotypes of fungi isolates were recovered. All 30 isolates were screened for P-solubilizing activity. 13 isolates (including 11 Penicillium and 2 Aspergillus) with remarkable P-solubilizing activity on National Botanical Research Institute’s phosphate growth (NBRIP) agar, as visualized by a clear zone around the colony, were selected. To identify the strain with the strongest P solubilizing ability, we analyzed the change in pH and the amount of solubilized P in NBRIP liquid medium. As shown in Fig. 1, strain 1107, which was identified as A. niger according to its colony morphology and microscopic characteristics, caused a decrease in pH from 7.0 on day 0 to pH 3.0 on day 10. After 10 d of culture, this strain had solubilized and released 689 mg P L–1 (Fig. 1).

2.2. Shelf-life of A. niger strain 1107 We evaluated the survival of A. niger strain 1107 in various carriers sterilized by autoclaving or gamma-ray irradiation and stored at 4 and 25°C, respectively for 7 mon. The survival of A. niger strain 1107 in various carriers sterilized using autoclaving or gamma-ray irradiation prior to storage at 4°C is shown in Fig. 2. During the first 5–6 mon of storage, most of the carriers (except composted cattle manure with 20% perlite, CCMP) sterilized by gamma-ray irradiation had higher inoculum loads than those in carriers sterilized by autoclaving. This effect was not observed after 7 mon of storage.

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of the biofertilizer on these carrier materials to colonize soil and promote plant growth. The objectives of this study were to select a suitable carrier for inoculating PSF, to confirm the sterilization effect of gamma-ray irradiation during biofertilizer preparation, and finally to develop an inexpensive and simple P-biofertilizer, which could be widely produced by most biofertilizer manufacturers in developing countries using currently available technologies. We evaluated the suitability of carriers consisting of materials that are widely available in developing countries, including corn cobs, wheat husks, and composted cattle manure mixed with perlite, in addition to peat. We compared the shelf-life of P-biofertilizer among the different carrier materials. The P-biofertilizer was inoculated into soil in a pot experiment. The survival of the fungus, its effects on available P in soil, and its effects on the growth of Chinese cabbage were evaluated. To investigate shelf life, we compared inoculants stored at room temperature (25°C) and at 4°C, respectively.

Solube P (mg L−1)

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Fig. 1 Changes in pH and soluble P concentration during fermentation in National Botanical Research Institute’s phosphate growth (NBRIP) liquid medium.

In the first 2 wk of storage, the number of viable cells increased dramatically in all of the carrier materials. The population of A. niger 1107 was higher than 7.1 log CFU spores g–1 inoculant for at least 7 mon at 4°C. This population size met the standard considered as acceptable in most countries (9 log CFU g–1 inoculant) (Ogbo 2010). The only exception was the population of A. niger in CCMP, which decreased from 7.8 to 4.5 log spores g 1 inoculant in 7 mon. Compared with CCMP, corn cobs with 20% perlite (CCP) and wheat husk with 20% perlite (WHP) were better materials to support the growth of, and maintain the population of, A. niger. The CCMP, CCP, and WHP carriers contained 1.2×107, 2.0×107, and 3.2×107 spores g–1 inoculant, respectively, after 7 mon. As shown in Fig. 3, the spore survival rate was higher in P-biofertilizer stored at 25°C than in that stored at 4°C during the first 1–3 mon, but after 3 mon, the survival rate was higher at 4°C than at 25°C.

2.3. Efficacy of biofertilizer We conducted a pot experiment to evaluate the ability of A. niger 1107 to colonize soil and its effects on plant growth. In all of the treatments, the A. niger 1107 population increased in the first 3 wk, and then decreased (Fig. 4). By 21 d after inoculation, the soil samples contained 1.1–6.6×106 spores of A. niger g–1 soil. As time went on, the population of A. niger 1107 decreased to 0.9–56.2×105 viable spores before plant harvesting. The most promising phosphate-solubilizing microorganisms (PSM) biofertilizer in the shelf-life experiment (that on WHP) showed the highest soil colonization rate (5.6×106 spores of A. niger g–1 soil), which was similar to that of the peat-based biofertilizer in soil. These results indicated that WHP is a good carrier material for biofertilizer. In contrast, composted cattle manure was not a suitable biofertilizer carrier, since the A. niger 1107

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The population of Aspergillus niger 1107 (log CFU g−1 inoculant)

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spores in this material showed the lowest soil colonization rate, even lower than that of free A. niger 1107 cells. The soil colonization experiment showed that the survival of

A. niger strain 1107 in peat, CCP, and WHP sterilized by gamma-ray irradiation was 3.87, 0.4, and 5.62 log CFU g–1 inoculant, respectively, and that in the same materials

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Fig. 4 Soil colonization of A. niger strain 1107 in various carriers sterilized by gamma-irradiation and autoclaving during plant growth. CCP, corn cob mixed with 20% of perlite; WHP, wheat husk mixed with 20% of perlite; CCMP, composted cattle manure mixed with 20% of perlite.

sterilized by autoclaving was 3.29, 0.2, and 3.16 log CFU g–1 inoculant, respectively. Therefore, the differences in the amount of A. niger 1107 spores among different materials were far greater than the difference in the amount of A. niger 1107 spores between the same carrier sterilized using two different methods. We evaluated the beneficial effects of P-biofertilizer on the growth of Chinese cabbage in a pot experiment. The effects of A. niger 1107 inoculations as both liquid and solid inoculants on the length of roots and shoots, wet weight, and dry weight are shown in Table 1. The root length and weight (including wet and dry mass) of plants inoculated with A. niger 1107 were significantly (P<0.05) higher

than those of uninoculated control plants. Treatments with A. niger 1107 on peat and WHP carriers were significantly more effective (P<0.05) for promoting plant growth than those treated with A. niger 1107 as free cells or on the CCMP carrier. The maximum increases in root length and dry weight (91.7 and 106.7%, respectively) were in plants treated with A. niger 1107 on gamma-irradiated peat. There were also very large increases in root length and dry weight (83.3 and 93.3%, respectively) in plants treated with A. niger 1107 on gamma-irradiated WHP. As shown in Table 1, after plant harvest, the amount of available P in soil treated with the A. niger 1107 was higher than that in non-inoculated soil. The P content in TCP-supplemented soil increased from 7.5 to 24.3 mg kg–1 after inoculation with the P-solubilizing fungus. The available P content differed significantly (P<0.05) among soils treated with biofertilizer on different carriers, but not between soils treated with biofertilizer on the same carrier sterilized using two different methods. The greatest increase in available P (a 1.6-fold increase) was in soil treated with A. niger 1107 on gamma-irradiated TCP.

3. Discussion In this work, A. niger strain 1107 showed a strong P-solubilizing ability (689 mg P L–1), which might be because of its strong ability to produce organic acids. Other studies reported that the ability of many fungi to solubilize phosphates in vitro is generally associated with the release of organic acids, which decreases the pH of the growth medium (EI-Azouni 2008; Ogbo 2010). As reported by Ogbo et al. (2010), there are wide variations among species in terms of the amount of soluble phosphate produced in liquid cultures (9.47–1 235 mg L–1). Even within the same fungal

Table 1 Effects of inoculation Aspergillus niger 1107 on growth and soil available P after harvest of Chinese cabbage plants under pot experiment Treatment1) Soil Soil+TCP Soil+TCP+1107 Soil+TCP+1107 in γ-irradiated peat Soil+TCP+1107 in autoclaved peat Soil+TCP+1107 in γ-irradiated CCP Soil+TCP+1107 in autoclaved CCP Soil+TCP+1107 in γ-irradiated WHP Soil+TCP+1107 in autoclaved WHP Soil+TCP+1107 in γ-irradiated CCMP Soil+TCP+1107 in autoclaved CCMP 1)

Shoot length (cm) 9.5±1.5 a 10.2±1.2 a 10.8±1.4 a 14.9±1.2 cd 14.6±1.0 bcd 13.5±0.8 bc 13.1±1.2 b 14.6±1.0 bcd 15.4±0.8 d 11.7±1.5 a 11.5±1.3 a

Root length (cm) 1.2±0.3 a 1.3±0.3 ab 1.7±0.2 bc 2.3±0.3 e 2.2±0.5 e 2.0±0.3 cde 1.8±0.4 bcd 2.1±0.3 de 2.2±0.5 e 1.7±0.3 bcd 1.6±0.2 abc

Plant weight (mg plant–1) 42.8±1.8 a 44.5±1.5 ab 54.5±1.9 bc 80.4±5.7 e 78.1±6.6 e 71.3±4.6 de 70.7±7.8 d 75.2±6.0 de 76.1±5.7 de 55.2±4.3 c 53.4±4.8 c

Dry mass (g plant–1) 4.5±0.6 a 4.6±0.6 a 5.8±0.2 b 9.3±0.8 e 9.1±1.2 e 8.0±0.7 cd 7.8±0.3 c 8.5±0.8 cde 8.7±0.4 de 6.2±0.3 b 6.0±0.5 b

Soil available P after harvest (mg kg–1) 14.9±0.9 a 17.5±1.5 b 19.4±0.7 c 24.3±0.7 f 23.8±1.0 f 20.5±0.9 cd 19.4±1.0 c 21.8±0.8 de 22.2±0.8 e 20.0±1.5 c 19.8±1.2 c

TCP, tri-calcium phosphate; CCP, corn cob mixed with 20% of perlite; WHP, wheat husk mixed with 20% of perlite; CCMP, composted cattle manure mixed with 20% of perlite. Results represent the mean of three plot (five plants in one plot) replicates±SD. Different letters show values that are significantly different (P<0.05) according to the Duncan’s test.

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species, the wide differences in experimental conditions used in various studies may explain some of the variations in P-solubilization ability, but the P-solubilization ability also varies markedly among different organisms and strains (Lapeyries et al. 1991). Previous works indicated that methods used to sterilize various carriers can substantially affect microorganism survival (Strijdom and Van Rensburg 1981; Khavazi et al. 2007). Similarly, Khavazi et al. (2007) reported that rhizobial populations were larger in materials pre-sterilized by gamma-ray irradiation than in those pre-sterilized by autoclaving. After 6 mon, however, this effect was only significant for a perlite/sugarcane bagasse mixture. In the shelf-life experiments, the survival of A. niger strain 1107 in the carrier materials sterilized by autoclaving was approximately 2 to 4 times higher than its survival in those sterilized by irradiation. This is probably because of changes in the chemical composition of the carriers during the autoclaving process (Strijdom and Van Rensburg 1981). Irradiation of the carrier material should not cause any physical and chemical changes to the inoculant, or cause it to produce toxins (Parker and Vincent 1981; Rizzuti et al. 1996; Daza et al. 2000). In the present study, the advantage of gamma-ray irradiation over autoclaving was not detected after 7 mon of storage. This may be because spore-forming or cyst-forming microorganisms were able to recover and replicate after sterilization with irradiation, and therefore, compete with the inoculant (Yardin et al. 2000). Gamma-ray irradiation is a good sterilization treatment because it is easy and it can be used on a large scale. However, it is important to note that gamma-ray sterilizers are not readily accessible in less developed countries. The storage temperature is another factor that affects the shelf-life of biofertilizers. Accinelli et al. (2009) reported a greater decline in A. flavus NRRL 30793 propagules at 25°C than at 4°C during a 6-mon storage period. Consistent with this, our results showed that room temperature was suitable for a short storage period, but 4°C was better for prolonging shelf-life during long-term storage. Generally, the biofertilizer on carriers promoted plant growth more effectively than did the free-cell biofertilizer. This is because carriers protect functional microbes from

soil or climatic stresses (Daza et al. 2000; Jain et al. 2010). Carrier materials may enhance the survival of inocula by providing microorganisms with a protective environment. This can allow them to survive in unfavorable conditions during the preservation and soil colonization process. In particular, once the microbe is introduced into soil, it must be able to survive in the subsurface zone to effectively solubilize P independently of the ecological conditions. There are many instances where different carrier materials have improved biofertilizer growth and survival. Finely ground peat is the most commonly used carrier in conventional inoculant production. However, peat is not always available, and the need to preserve wetland ecosystems makes the extraction of peat inadvisable in some areas. Thus, there is a need to identify other materials that support good growth and survival of microorganisms. Agro-wastes are good alternatives to peat as carrier materials. Ogbo (2010) used cassava wastes as carriers for two fungi, A. fumigatus and A. niger. In that study, the ground cassava waste met many of the criteria for a carrier material, and it supported the growth of the studied organisms. Rivera-Cruz et al. (2008) used poultry manure and banana waste as the inoculant carrier for P-solubilizing bacteria. They found that the application of these biofertilizers improved plant performance and the physical and microbiological properties of the soil. Perlite has been used as a carrier for Rhizobium leguminosarum bv. phaseoli, R. tropici, Bradyrhizobium japonicum, and Bacillus megaterium inoculants. The viable colony counts of these bacteria remained stable at 107–109 g–1 for at least 180 d when the carrier was stored at 4 or 28°C (Daza et al. 2000). Six different mixtures of perlite and charcoal/sugarcane bagasse were evaluated as carriers for Bradyrhizobium japonicum, and all of them supported rhizobial growth over a 6-mon period (Khavazi et al. 2007). In the present study, populations of the P-solubilizer A. niger remained stable in sterilized WHP for at least 7 mon. This is because of its high nutrient content, high water holding capacity, and good aeration characteristics (Table 2), which are three key characteristics of good carriers according to Smith (1992). Our results showed that composted cattle manure was not suitable as biofertilizer carrier to enhance the microbial population. This might be

Table 2 Chemical and physical characteristics of materials used as carriers Material Peat Wheat husk Corn cob Composted cattle manure 1)

EC, electrical conductivity. OM, organic matter. 3) WHC, water holding capacity. 2)

N (%) 1.73 0.42 0.35 1.63

P (%) 0.21 0.15 0.06 1.03

K (%) 0.32 1.14 0.91 1.78

EC (ds m–1)1) 0.24 2.08 1.07 24.5

pH 6.14 5.43 5.64 5.50

OM (%)2) 65.4 31.5 89.4 46.2

WHC (%)3) 100 300 420 120

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because of its higher soluble salt content (EC 24.5 dS m–1), which would affect major microbial processes including respiration and ammonification. It has been reported that salt-tolerant fungi, actinomycetes, and a few bacteria can survive at higher salt concentrations (Rynk 1992; EI-Azouni 2008). In general, EC values between 0 and 3.5 dS m–1 are acceptable for general crop growth; at EC values exceeding that range, the salt concentration will hinder plant growth by affecting the soil-water balance (Naidu et al. 2010). As shown in Fig. 4, the soil colonization experiment showed that the differences in the survival counts of A. niger strain 1107 among the various carriers sterilized by different methods ranged from 117 to 235%. In contrast, the differences in survival counts among the various carrier materials ranged from 145 to 6 244%. It is noteworthy that compared with the complex soil environment, the effect of the sterilization method on the viability of A. niger strain 1107 spores during plant growth was insignificant. This result indicated that the changes to the carriers resulting from the sterilization procedure were negligible. The wide range in survival counts among the different carrier materials showed that some materials were much more suitable than others for survival of A. niger strain 1107. After 60 d incubation, the highest survival count was in peat, followed by WHP. Consistent with our findings, inoculation with P solubilizers has been reported to increase the soil available P content (Mittal et al. 2008; Jain et al. 2010; Jain et al. 2012). The effects of A. niger 1107 to promote plant growth may be related to the increase in available phosphate in soil. This would also benefit the next crop, as more available phosphate in soil would increase its fertility (Jain et al. 2010). In summary, although WHP was not superior to peat as a carrier, it is a suitable alternative carrier to peat for the P-solubilizing strain of A. niger. These results are important because the demand for biofertilizer in China is greatly increasing, but biotechnological resources, especially carrier materials for biofertilizer, are limited.

4. Conclusion According to the shelf-life experiment, gamma-ray irradiation is suitable as a simple sterilization method that can be used to treat large amounts of material. The most effective carrier material for growth and maintenance of PSF populations was WHP; there was a higher rate of conidia survival on this material than on other materials, and when it was applied as a biofertilizer to Chinese cabbage, it beneficially affected plant yield and increased the soil available P concentration. The wide application of P-biofertilizer as an alternative to chemical fertilizers in developing countries will contribute to lower-input farming systems and a cleaner environment.

5. Materials and methods 5.1. PSM isolation and identification More than 20 rhizospheric soil samples were collected from various farmlands in Beijing, Hebei Province, and Heilongjiang Province, China. For each sample, 1 g soil was suspended in 10 mL sterilized water, diluted to 10–7, and then 100 µL of each dilution was spread on the potato-dextrose agar (PDA) medium. After incubation of the plates for 4 d at 30°C, colonies on the plates were selected, purified by repeated culturing, and maintained on PDA slants at 4°C. Preliminary screening for phosphate solubilization was conducted by a plate assay method using modified National Botanical Research Institute’s phosphate growth (NBRIP) agar medium (pH 7.0) (Nautiyal 1999). The medium contained (in g L–1): glucose, 10.0; Ca3(PO3)2, 5.0; (NH4)2SO4, 0.5; NaCl, 0.3; KCl, 0.3; MgSO4·7H2O, 0.3; FeSO4·7H2O, 0.03; MnSO4·4H2O, 0.03; and agar, 18. Fungal strains were pin-point inoculated onto plates containing solidified NBRIP medium under aseptic conditions. The plates were incubated at (28±2)°C for 7 d and the diameter of fungal colonies was continuously observed. The fungal colonies surrounded by a clear halo zone showed P-solubilization activity, and were selected for further tests. These strains were maintained on PDA slants at 4°C. To confirm the P-solubilizing activities of the selected strains, each strain was incubated in 100 mL NBRIP liquid medium at 30°C and 120 r min–1 on a rotary incubator shaker for 10 d, and then the amount of P released was measured by the molybdenumblue method (Murphy and Riley 1962). At the same time, pH was recorded using electrode pH meter (Mettler Toledo Delta 320).

5.2. Carrier preparation Carrier materials were first powdered and passed through a 100-mesh sieve before physicochemical characterization (Table 2). The main criteria used to select carrier materials were the ability to adjust the pH to neutral (pH 7.0), high water holding capacity, low cost, and wide availability. The pH of all of the materials was adjusted to pH 7.0 with CaCO3 before use. To adjust pH, materials were thoroughly mixed with CaCO3 powder. Four carriers: (1) peat, (2) corn cob mixed with 20% perlite (CCP), (3) wheat husk mixed with 20% perlite (WHP), and (4) composted cattle manure mixed with 20% perlite (CCMP) were evaluated.

5.3. Carrier sterilization For each of the four carriers, 500 g of carrier material was

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placed into each of 10 cotton bags (50 cm×16 cm). The cotton bags were approximately 1.0 mm thick. These packages were sterilized by gamma-ray irradiation or autoclaving. For irradiation, the packages were placed in 0.08-mmthick polypropylene plastic bags. Gamma-ray irradiation was carried out using a 60Co gamma cell source. Irradiation was applied at a dose of 50 kGy at a rate of 15 Gy min–1. For the autoclaving treatment, the samples were autoclaved for 40 min at 121°C. The sterilized bags were placed into another large sterile cotton bag after cooling overnight in the autoclave. After sterilization, the packages were dried for 12 h at 60°C in a blow-type oven.

5.4. Inoculant preparation and incubation To obtain fresh spores of strain 1107, the fungus was grown on NBRIP agar medium at 30°C for 5 d. The spores were recovered from plates in 5 mL sterile 0.2% Tween-20 collected by gentle scraping. The concentration of spores was determined by microscopic counts using a hemocytometer. The spore density was adjusted to a final concentration of 108 spores mL–1. The spore suspension was injected aseptically into the sterilized carriers to 40% of water holding capacity using a sterile syringe injector. The bags were then thoroughly kneaded and incubated at 30°C for 2 wk before the storage experiment. The population size of strain 1107 in the carriers was measured by the plate count method.

5.5. Determination of shelf-life To evaluate shelf-life, the formulated products were transferred into sterilized screw-top tubes (50-mL volume) and stored in the dark at 4 and 25°C. The biofertilizer was turned over every 3-4 d for 7 mon. Non-inoculated raw material was used as the control. After 7, 14, 30, 60, 90, 120, 150, 180, 210 d of storage, the amount of surviving fungus (spores) was evaluated by the plate count method. Aseptic conditions were maintained throughout this process.

5.6. Pot experiment Black soil (top soil, 0–20 cm) was collected from an arable field in Heilongjiang Province, China. Air-dried soil samples were ground and homogenized by passing through a 100mesh sieve after removing stones, soil fauna, and plant debris. The soil properties were as follows (g kg–1 air-dry soil): sand (0.05–0.2 mm), 280; silt (0.002–0.05 mm), 450; clay (<0.002 mm), 270; organic carbon, 23.03; total N, 1.28; available P 0.02; and pH 5.7 (1:2.5 soil/water mixture).

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A mixture of 0.5 g tri-calcium phosphate (TCP) per kilogram soil was prepared and placed in plastic pots (height, 13 cm; upper diameter, 12 cm; lower diameter, 9 cm). The pots with different treatments were arranged in a randomized complete block design with triplicates of each treatment. There were 11 treatments in total: T1: soil; T2: soil+TCP; T3: soil+TCP+ A. niger 1107; T4: soil+TCP+A. niger 1107 in peat sterilized by gamma-ray irradiation; T5: soil+TCP+A. niger 1107 in peat sterilized by autoclaving; T6: soil+TCP+A. niger 1107 in CCP sterilized by gamma-ray irradiation; T7: soil+TCP+A. niger 1107 in CCP sterilized by autoclaving; T8: soil+TCP+A. niger 1107 in WHP sterilized by gamma-ray irradiation; T9: soil+TCP+ A. niger 1107 in WHP sterilized by autoclaving; T10: soil+TCP+ A. niger 1107 in CCMP sterilized by gamma-ray irradiation; T11: soil+TCP+A. niger 1107 in CCMP sterilized by autoclaving. Chinese cabbage seeds were surface sterilized by immersion in 0.1% sodium hypochlorite solution for 10 min. The seeds were washed three times with distilled water and then planted in pots (9 seeds pot–1, with equal distance between seeds). Then, 10 mL spore suspension or 10 g prepared biofertilizer was applied uniformly to seeds, which were then covered with a 20-mm-thick soil layer. After 1 h, the pots were sprinkled with water. The pots were irrigated periodically to maintain soil moisture at between 30 and 50%. At 1 wk after germination, the seedlings were thinned to five per pot. Plants were harvested at 60 d after sowing, and their shoot and root length and dry mass were recorded. The dry weight of plant tissues was determined after drying at 75°C. After harvesting, the available P concentration in pot soil was analyzed by the NaHCO3-extractable phosphorus colorimetric method (Olsen et al. 1954). At various time points (7, 14, 21, 28, 35, 42, 60 d), triplicate soil samples around the seed were collected and the A. niger 1107 colonization rate was estimated by the plate count method.

5.7. Statistical analysis All experiments were conducted in triplicate and data were subjected to analysis of variance. Mean values were compared using one-way ANOVA Duncan’s test and significant differences were detected at the P=0.05 level. The number of fungal spores derived from soil or inoculant is expressed as the log(10) transformed colony forming units (CFU) g–1 weight.

Acknowledgements This work was financially supported by the Special Fund for Agro-Scientific Research in the Public Interest, China (201003014) and the Central Public-Interest Scientific

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Institution Basal Research Fund, China (202-27).

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