Evaluating the effect of gamma irradiation and steam sterilization on the survival and growth of composted sawdust fungi in Ghana.

The growth and survival of some fungi associated with composted and pretreated sawdust particularly for mushroom cultivation were investigated on two growth media; Cooke’s and Oxytetracycline Glucose Yeast Extract (OGYE). Some fungi were isolated during the composting of sawdust over a period of 28 days as well as after pretreatment with gamma irradiation doses of 5, 10, 15, 20, 25 and 32 kGy and moist heat of 100±2oC for 2.5 hours. Fungal counts ranged 4.725.77 log10 CFU/g and 3.44.1 log10 CFU/g respectively for both media. Both pretreatment methods effectively reduced (p<0.05) fungal counts by an average of 1.48 (irradiation) and 2.22 (steam) log-cycle reductions on OGYE while there was an average of 3.13 (irradiation) and 1.10 (steam) Original Research Article Kortei et al.; BMRJ, 7(4): 180-192, 2015; Article no.BMRJ.2015.110 181 log-cycle reduction on Cooke’s. Corresponding radiation sensitivities (D10 values) of 5.94±2.06 kGy and 5.64±1.12 kGy were recorded for fungi on both media respectively. Five species belonging to three genera were isolated on OGYE and among the fungi were Aspergillus niger, Aspergillus fumigatus, Aspergillus ustus, Mucor racemosus and Rhizopus stolonifer. Ten species belonging to four genera were also isolated on Cooke’s medium included Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus ustus, Aspergillus terreus, Aspergillus parasiticus, Aspergillus alutaceus, Rhizopus stolonifer, Fusarium oxysporum and Mucor racemosus. There was an observed reduction (p<0.05) in species diversity after pretreatment. The presence of these microorganisms on sawdust also has serious repercussions on animal and human health.


INTRODUCTION
Sawdust which is a source of organic waste material is used extensively in horticulture, in soil amelioration, as a substrate for cultivation of mushrooms and in diverse applications. Nonetheless, sawdust attains its optimum utilization if it is well decomposed and the lignin content turned to humus. Improperly decomposed sawdust could result in nitrogen immobilization and produce some toxic substances which will affect plants, fungi, animals and humans [1].
Composting is a process of exothermic biological oxidation of various organic wastes in the presence of air involving microorganisms. Through microbial decomposition, the organic matter is stabilized, matured and deodorized into a product rich in humic substances that can be used as organic soil conditioner, easy to store and distribute [2,3]. Modern concept of environmental management relies on the recycling of wastes. In this perspective, composting appears to be a safe form of treatment of some wastes and the reclamation of the nutrients contained in them [4]. It is environmentally friendly because the process produces a marketable end-product that can be used as a soil conditioner and organic fertilizer. Composting is therefore regarded as a successful strategy for the sustainable recycling of organic wastes [5,6].
The active component mediating the biodegradation and conversion processes during composting is the resident microbial community, among which fungi play a vital role [7]. Therefore, optimization of compost quality is directly linked to the composition and succession of microbial communities in the composting process [8,9]. There is practically no substance existing in nature that is not used by one microorganism or another [10]. It is therefore necessary to identify the microorganisms present in the different processes, as several different species of microbes are usually involved [11,12]. Microbes play key role in the process monitoring of microbial succession is important for the effective management of the composting process.
Since composting methods and different substrates are associated with different composition of a microbial community, monitoring of the resident microbial population in compost is essential to determine its quality and field of application [8,13].
Fungi use many carbon sources; mainly lignocellulosic polymers and can survive in extreme conditions. They mainly are responsible for compost maturation [14]. They also degrade complex polymers such as polyaromatic compounds or plastics and are being increasingly applied to bioremediate soils contaminated with a wide range of pollutants [15,16].
Steam pretreatment of sawdust for mushroom cultivation, is the most widely used method in Ghana [17,18]. It has become essential to explore other methods of pasteurization and improve efficiency of production. Gamma irradiations which have short wave length, high energy photons, and have deep penetrating power so could serve both as a decontaminating agent and a hydrolytic agent [19,20,21] for the bioconversion of lignocellulosic materials to produce mushrooms [21].
The objective of this paper is to investigate the fungal populations and species diversity associated with the composting process, gamma irradiated and steam / moist heat pretreated sawdust substrate principally for mushroom cultivation.

Composting
The compost was prepared by the outdoor single phase solid waste fermentation. Fresh sawdust obtained from Timber market Accra was mixed and composted as described by [21]. The compost was then stacked into a heap of about 1.5 m high, 1.5 m long and 1.5 m wide. This heap was left for composting for 28 days with regular turning every 4 days. At weekly intervals samples, moisture content of the compost were adjusted to approximately 68 -70% [22] and then supplemented with rice bran (12%) and lime (0.5%).

Enumeration and Characterization of Mycoflora
The dilution plate technique was used in estimating fungal populations. About 10 g fresh weight of sample was placed in 250 ml Erlenmeyer flask containing 100 ml sterile distilled water. The mixture was shaken at 140 rev. /min in a Gallenkamp Orbital Shaker for 30 min. Aliquot (1ml) of the suspension was placed in sterile universal bottles (MaCartney tubes) containing 9 ml of 0.1% peptone, and was serially diluted up to 1:10 3 . The fungal population was enumerated on modified Cooke's medium [23] and OGYE incubated at 30-32ºC for 3-5 days for species diversity. The fungi were identified using their microscopic, morphological and cultural characteristics as outlined by [24].

Determination of Occurrence Percentage of Fungi
The incidence of occurrence of the different fungal isolates was done by a modified formular of [25,26]. The frequency of occurrence of the fungal pathogens from the sawdust was determined. The total number of each isolate in the sawdust sample was obtained against the total number of all the isolates in the sample screened.
The mean value of this gives the occurrence percentage as the following equation shows: occurrence percentage = X/N × 100 (1) where X = total number of each isolate in the sample and N = total number of all the isolates in the sample.

D 10 Values Determination
The D 10 value is the reciprocal of the slope of the exponential part of a survival curve. This value may also be obtained from equation (2). The data was subjected to regression analysis. The surviving fractions, log 10 (N/N 0 ) of microorganisms, was calculated and used as relative changes of their actual viable cell counts. The D 10 values were calculated by plotting log 10 (N/N 0 ) against dose (D) according to the equation D 10 = Radiation Dose (D) log 10 (No-N) (2) where No is the initial viable count; N is the viable count after irradiation with dose D; D is the radiation dose [27,28,29]. The linear correlation coefficient (r 2 ) and the regression equations were also calculated.

Statistical Analysis
The values obtained for the fungal counts were done in the standard forms and then transformed to logarithmic values and subjected to analysis of variance (one way ANOVA) using SPSS (Illinois, USA) version 9 for Microsoft windows.

Fungal Population during Composting
The dynamics of fungal community may be attributed generally to abiotic variables and nature of substrate [30]. There was variation of the fungal species in the sawdust with respect to the period of composting. The total mycoflora population enumerated on Cooke's medium was comparatively higher than on OGYE. The highest mycofloral population of 5.77 log 10 CFU/g was recorded for 28 th day of composting while the lowest population of 4.72 log 10 CFU/g for the 4 th day of composting. Statistically, mycoflora population of 0 (first), 4 th , 8 th and 24 th days of composting which corresponded to 5.00, 4.72, 4.98 and 5.00 log 10 CFU/g respectively differed significantly (p<0.05) from 12 th , 16 th , 20 th and 28 th days of composting which corresponded to 5.74, 5.69, 5.51 and 5.77 log 10 CFU/g (Fig. 1). However, the highest mycoflora population enumerated on OGYE was 3.8 log 10 CFU/g for 0, 12 th , 28 th days while the lowest recorded was 3.4 log 10 CFU/g for 24 th day of composting. Composting days 0, 4, 12, 28 and 8 corresponding to 3.8, 3.7, 3.8, 3.8 and 4.1 log 10 CFU/g respectively differed significantly (p<0.05) from days 16 th and 20 th corresponding to 3.6 and 3.5 log 10 CFU/g respectively which differed significantly (p<0.05) from 24 th day of 4.1 log 10 CFU/g ( Fig. 1).
Generally, at the beginning of composting, there was a comparatively low mycofloral population which suggested a minimal occurrence of factors such as moisture content, relative humidity and a wide fluctuation in air temperature which did not favour the sporulation of fungi on the sawdust substrate [31,32]. Microbial activities however increased as the composting period progressed. There was an increase in temperature, moisture content, pH, relative humidity and electrical conductivity of the activities according to [7] resulted in an increase in metabolic activities to produce enzymes by resident microorganisms to decompose the lignocellulosic materials. The enzymatic activities in compost piles are effective indicators for stress or adaptive practices of the microorganism to different environmental conditions, particularly to feed stock sources. Various hydrolytic enzymes can control the rate of decomposition of complex polymers during composting [33]. Obodai et al. [34] also suggested that the environmental and nutritional conditions created during composting might have selectively favored certain fungi to the detriment of others. This trend was in agreement with works of some researchers [34,35].

Effect of Gamma Irradiation and Steam Sterilization on Mycofloral Population of Sawdust Substrate
Both irradiation and steam techniques of pretreatment were effective in reducing the mycofloral population of sawdust. Although Comparatively high fungal counts were recorded on Cooke's medium than on OGYE medium (Fig.  2), gamma radiation dose of 5 kGy reduced the initial mycoflora of 5.77 log 10 CFU/g (control) by 0.68 log cycles to 4.89 log 10 CFU/g which did not differ (p>0.05) from the control. Doses 10 and 15 kGy recorded log cycle reductions of 1.97 and

Occurrence Percentage of Microorganisms during Composting of Sawdust Substrate
Various mycoflora and microflora are involved in the composting of agricultural wastes and these range from fungi and bacteria that are thermophiles due to the temperature rise of 50-60ºC, mesophiles and tolerant mesophiles.

Cooke's OGYE
Results obtained imply a relatively high numbers of microorganisms were involved in the decomposition of the T. scleroxylon sawdust compost making it suitable for the growth of mushrooms as the diversity encourages different contributions biologically, chemically or physically [37,38].
The genus Aspergillus was predominant in all composting days of sawdust substrate. Several members of this genus have been reported by several researchers [39,40,41,42,43,44,45] to be capable of hydrolyzing the β-(1-4)-glucosidic linkage in the cellulose chain. According to [7], there is the production of metabolites in the compost during fungal growth which penetrates a cell and inhibits activity by chemical toxicity. Antagonism among fungi may be in the form of competition for nutrients, chemical antibiosis and lysis of mycelium. Antibiosis is the inhibition of one generation by the metabolic product of another. Although it is usually an inhibition of growth and sporulation, it may be toxic. Lysis is destruction and decomposition of biological materials by enzymes of the parasite. Fungal phenology observed in the compost may be partly attributed to antibiosis and lysis of mycelium.
Fungal species occurrence was irregular probably because the environmental and nutritional conditions created during composting selectively favored certain fungi to the detriment of others.
Results obtained agreed with similar works reported by some researchers [34,46] as they investigated the various fungi associated with the composting of sawdust for mushroom cultivation.   chemically changed and the phosphodiester backbone is broken (single-strand breaks) but some 5-10% double-strand breaks also occur [47]. Aquino [48] reported that radiation-resistant organisms are capable of withstanding effects on the plasma membrane of the cells. The rate of dose absorption has no effect on survival except when oxygen replenishment is involved. Elevated temperatures above 45°C are synergistically bactericidal because the higher temperatures damage the repair systems. To achieve this effect in spores, temperatures of 80 -98°C are needed. Subfreezing temperatures raise the radiation resistance of vegetative cells as the water activity decreases and the diffusion of radicals is restricted.

Radiation Sensitivity (D 10 Values) of Fungi on Sawdust
Radiation sensitivity (the killing effect of radiation) in microorganisms is generally expressed by the decimal reduction dose or D 10 value [27] which was calculated from equation (1). The relative sensitivity of different microorganisms to ionizing radiation is based on their respective D 10 values (which is the dose required to reduce the population by 90%). Lower D 10 values indicate greater sensitivity of the organism in question. The data in Table 1.
shows that gamma radiation doses achieved significant reduction in mycoflora during pasteurization of sawdust for mushroom production. On OGYE there was a good correlation coefficient of 0.971 with a corresponding mean D 10 value of 5.94±1.18 kGy (Fig. 7). Also on Cooke's medium, a good correlation coefficient of 0.924 was established with a corresponding mean D 10 value of 5.64±1.12 kGy was obtained (Fig. 7). This implies that a slightly higher mean dose of 5.94 kGy was required to inactivate fungi of composted sawdust. Hossain et al.

pH
The pH of fermenting substrate, initially recorded 7.39 which was neutral (Table 2). However, pH fluctuated to basic / alkaline (7-9) and then to acidic (6.81-8.04) as composting time increased. The drop in pH could probably be due to rapid breakdown of soluble and easily degradable carbon sources, resulting in a pH drop due to organic acids formation [52]. According to Tchobanoglous et al.
[53], initial pH values of 7-7.5 is recommended as it aids production of lactic acid and acetic acids during initial degradation of biomass. Interestingly, at the 20 th day of composting the pH value 9.17 was recorded which is in agreement with some researchers [54,55] who stated that in the thermophilic stage of composting, the pH could rise to 9 resulting in the release of ammonia, and there after the pH returns to near neutral conditions as the compost become mature. After pretreatment, pH ranged 7.45-8.39 (Table 3).

Moisture Content
Moisture content during the composting period ranged between 58.2 -65.8%. Moisture is important in composting processes for two reasons: it facilitates substrate decomposition through mobilizing microorganism activities and also provides better conditions for nitrogen fixation in the compost. A low moisture content below a critical level (<30%), would decrease activities of microorganisms by restricting the motility and make them dormant [56]. Under drier conditions, the ammonium and ammonia present generate a higher vapor pressure; thus conditions are more favorable for nitrogen loss. On the other hand, a moisture content which is too high (>65%) could cause oxygen depletion and losses of nutrients through leaching [57].