Gamma Irradiation and Autoclave Sterilization Peat and Compost as the Carrier for Rhizobial Inoculant Production

Rhizobium is a biofertilizer for leguminous crops. To formulate this form of fertilizer, the suitable sterilization processes of carrier are important. Therefore, the aim of this research was to elucidate the process of gamma irradiation and autoclaving on peat and compost based carriers for rhizobial inoculant production. Carriers with 10% moisture content packing in polyethylene bag could be efficiently sterilized by irradiation at 10-20 kGy, or by autoclaving with tyndallization approach (autoclaving two times in a row at 121oC for 60 min, with waiting period of 18 hours after each time of autoclaving). The number of Bradyrhizobium sp. PRC008 was in the range of 10-10 cfu/g in both irradiated and autoclaved peat after 6 months storage. However, the numbers of bradyrhizobial cell were reduced in compost sterilized by both methods after one month storage. These results indicated that carrier material had an important influence on inoculant quality, while sterilization processes using gamma irradiation and autoclaving with tyndallization approach could be used for efficient rhizobial inoculant production with peat based carrier.


Introduction
Using rhizobial inoculant is a clean technology for sustainable agriculture.Rhizobial inoculants have been used as an environmental friendly source of nitrogen fertilizer for several decades to reduce putting chemical nitrogen fertilizer into the soil, as well as the cost of legume production.There are several forms of rhizobial inoculant available in the market, including solid or liquid forms that can maintain the survival of effective rhizobia at the level of 10 8 cells/g for at least 6 months (Stephens & Rask, 2000).Even though the liquid form of rhizobial inoculants is applied in the production process more easily than solid form, the survival of rhizobial cell in liquid inoculants depends on strain of rhizobia and polymeric additive substance incorporated into liquid inoculants formulation (Tittabutr et al., 2007).Thus, solid inoculants, especially those using peat-based carrier, are still popular for biofertilizer inoculant production because peat could support the survival of bacteria in long term storage (Kishore et al., 2005;Okon & Labandera-Gonzalez, 1994).However, peat is limited in many countries including Thailand, so it is necessary to find carriers that are locally available for commercial scale production.The appropriate material to be used as a carrier in rhizobial or biofertilizer inoculant production should be non-toxic material, have good water holding capacity, support bacterial growth and survival, be easily prepared in powder form, and have nearly neutral pH (Albareda et al., 2008;Khavazi et al., 2007;Smith, 1992).Several materials have been tested as an alternative carrier source for rhizobial inculants, such as soil, coal, vermiculite, perlite, attapulgite, sepiolite, amorphous silica, cork compost, grape bagasse, and other plant composts (Albareda et al., 2008;Ferreira & Castro, 2005;Khavazi et al., 2007).However, the quality of inoculant is varied according to the physicochemical and biological properties of material as well as the sterilization method applied to carrier (Khavazi et al., 2007;Swelim et al., 2010).Since contaminant microorganism is the main problem that affects the quality and shelf-life of rhizobial inoculant, sterilized carrier is necessary to be accomplished prior injection of the pure culture of rhizobia into carrier.
Commercial rhizobial carriers are normally sterilized by gamma irradiation or autoclaving, depending on the accessibility and availability of instrument.Mechanism of sterilization by autoclaving is based on the wet killing of microorganisms under high temperature (121ºC) and high pressure (15 ponds/inch 2 ) for a period of time, depending on the size and the composition of material.However, the problem of spore forming microorganisms contaminant remains in the carrier after autoclaving is the main problem of using this sterilization method.While the mechanism of sterilization by gamma irradiation is due to the direct breakdown of double strand DNA, or occurring from the ionized water molecules form free radicals and disrupt biological system in the cell (Hansen & Shaffer, 2001).Sterilization of rhizobial carrier should consider in the physical and chemical changes of material which should not produce or release toxic substances and not destroy the available nutrients in material (Strijdom & van Rensburg, 1981).Another consideration is the efficiency of sterilization method, since high numbers of contaminating microorganisms significantly reduce the quality of rhizobial inoculant.Although gamma irradiation at high dose rate is more effective than autoclaving, the number of rhizobia in carrier is not significantly different after 6 months storage (Khavazi et al., 2007).The dose required to sterilize carrier varies with the property of material, the density, moisture content, initial contaminating load and packaging configuration.However, higher dose rate of gamma rays may produce higher toxic breakdown product from the carrier material, and some of spore forming microorganisms are still found when storing the carrier for a period of time (Yardin et al., 2000).Although the dose rate more than 50 kGy is usually used for efficient sterilization of carrier, lower dose rate is required for reducing energy and time of sterilization as well as reduce the toxic substances that may be produced when using high dose of gamma irradiation.This study aimed to find out the process of carrier preparation to achieve lowest dose rate that effectively sterilized carrier by gamma irradiation.The factors of carrier moisture content and plastic type container were elucidated.As well as the strategy of killing geminated spore forming microorganism by the application of tyndallization approach with autoclaving sterilization was also determined in order to minimize the contaminating microorganisms in the compost and peat for using as rhizobial carrier in inoculant production.

Carrier Materials
Two types of carrier, peat and compost, were used throughout the experiments.Peat was derived from Department of Agriculture (DOA), Thailand, while compost was obtained from Suranaree University of Technology.Compost was made from the mixture of agricultural wastes, such as cassava peel, filter cake, chicken dung and cow dung.The pH of peat and compost was 4.5 and 7.49, respectively.Both peat and compost were first milled and passed through a 100-mesh sieve, before the pH of peat was adjusted to nearly 7.0 by using CaCO 3 .Peat and compost were sent to Soil Laboratory, School of Plant Production Technology, Suranaree Univeristy of Technology, Thailand to determine the physical and chemical characteristics of carriers according to the standard method.

Carrier Preparations
The moisture contents of carrier were adjusted to 10, 20, and 30% by using water, and 80 g of each carrier were packed into 5×8 (inches×inches) plastic bag, either using the polyethylene (PE) or polypropylene (PP) bags with a thickness of 0.08 mm prior to sterilization.Each package had thickness of 0.8 cm.The moisture content of carrier and type of plastic bag container were varied according to each experiment.

Sterilization by Gamma Irradiation
Carrier packages were sterilized with gamma irradiation at Thailand Irradiation Center, Thailand Institute of Nuclear Technology (Nakhon Na-yok, Thailand).Cobolt-60 was used as a source of radiation.The packages were sterilized with different doses of gamma irradiation at 5, 10, 15, 20, and 25 kGy.The dosage received at various depths and locations were measured by using dosimeter to ensure the amount of radiation doses.The gamma irradiated packages were delivered to Suranaree University of Technology within a week for determination of the number of microbial contaminants in carrier after gamma sterilization.

Sterilization by Autoclaving
Carriers were moistened to 10% before packing 80 g of each carrier into 5×8 (inches×inches) of PE bag.The tyndallization approach was applied for carrier sterilization.The autoclave was operated for 2 times in a row at 121ºC for 60 min, while the waiting period after each time of autoclaving was varied at 18 and 24 h.

Microbial Contaminants Enumeration
The number of microbial contaminants in sterilized carrier package was determined at 1 week after sterilization by aseptically removing 10 g of carrier from each bag, and carrier was diluted in 90 ml sterilized water, and shaken at 200 rpm for 30 min.The sample was 10-fold serial diluted in sterilized water and plated in duplicate onto plate count agar (PCA) and potato dextrose agar (PDA) for bacteria and molds enumeration, respectively.Colonies were counted after incubation at 28ºC for 5 days, and calculated as log number of colony forming unit (cfu)/g dry weight carrier.The torn carrier packages were excluded from the experiment.

Inoculant Preparation and Storage
Bradyrhizobium sp.PRC008, obtained from DOA, Thailand has normally been used as an inoculum strain for inoculant production by DOA.This strain was recommended to use with mungbean (Vigna radiata L.) to reduce the use of chemical nitrogen fertilizer.In this study, PRC008 was cultured in a yeast extract mannitol (YEM) broth until late log phase.The culture was diluted 10 times, and 20 ml of diluted culture were aseptically injected into the bag containing 80 g of sterilized carrier with 20% of moisture content.Bags were thoroughly kneaded by hands, and the final moisture content of carrier was 40% at this step.Then, bags were incubated at 28ºC for 1 week and then left at room temperature (28-30ºC) for 6 months.

Enumeration of Rhizobial Cells by Plant Infection Count
The number of effective Bradyrhizobium sp.PRC008 was determined every month for 6 months after the injection by using plant infection count based on the principle assumptions underlying the most-probable-number (MPN) method (Somasegaran & Hoben, 1994).

Physicochemical Characteristics of Compost
In order to determine whether the compost had similar properties to peat, the physical and chemical characteristics of compost and peat were determined.The results are shown in Table 1.Peat and compost were much different in pH, organic matter, nitrogen, and phosphorus contents.The pH of compost was readily in neutral range, which would be suitable for survival of rhizobia, while acidity of peat must be neutralized by using CaCO 3 .The organic matter of peat was 4.4 times higher than in the compost.The amount of organic matter may contribute to longer survival of rhizobial cells in the carrier.Khavazi et al. (2007) reported that the mixing of perlite with high organic matter containing materials, such as sugarcane bagasse or malt residue could increase the number of bradyrhizobial cell survival at six months storage.Although the organic matter of compost was lower than peat, compost had higher nutrient contents.The high nutrient content was one of the key characteristics of good carrier (Smith, 1992).Compost in this study was made from the mixture of cassava peel, filter cake, chicken dung, and cow dung.However, not all sources of compost material could be used as carrier.Some compost materials, such as grape bagasse, result in a sharp decline of the population of Ensifer (Sinorhizobium) fredii SMH12 and Bradyrhizobium japonicum USDA110 after 15 days storage, probably because high content of phenolic compounds in the compost affects the growth of rhizobial cells (Albareda et al., 2008).Although compost used in this study has several characteristics that indicate its potential as a good carrier for rhizobial inoculant production, the final decision is based on rhizobial cell survival during storage.Thus, the appropriate sterilization methods using gamma irradiation and autoclaving with tyndallization approach, as well as rhizobial survival test in the compost as carrier, were elucidated below by comparing with peat carrier.Peat was adjusted to pH 7.0 by using CaCO 3 before using in the experiments.

Factors Affecting the Sterilization Efficiency by Using Gamma Irradiation
Gamma irradiation is a technique used for sterilization of several products, such as medical and pharmaceutical products, agricultural products, food products or food packages.However, sterilization using gamma irradiation with peat or compost, which have complex carbon-based structure and usually contain high level of contaminating load need to determine the appropriate dose rate and other factors that influence sterilization efficiency.The factor of material moisture contents (10, 20, and 30%) and types of plastic container (PP and PE) were examined in order to use lowest dose rate of gamma radiation for sterilization.Due to the limited amount of peat in this experiment, only compost was used to determine the appropriate moisture content of material and packed in PP bag before

Table 1 .
Physical and chemical properties of peat and compost