Effect of Corynebacterium glutamicum on Livestock Material Burial Treatment

Abstract

In recent years, foot-and-mouth disease has occurred in all parts of the world. The animals with the disease are buried in the ground; therefore, their concentration could affect ground or groundwater. Moreover, the complete degradation of carcasses is not a certainty, and their disposal is important to prevent humans, livestock, and the environment from being affected with the disease. The treatment of Corynebacterium glutamicum is a feasible method to reduce the risk of carcass decomposition affecting humans or the environment. Therefore, this study aimed to investigate the effect of C. glutamicum on the soil environment with a carcass. The composition of amino acids in the soil treated with C. glutamicum was generally higher than those in the untreated soil. Moreover, the plant root in the soil samples treated with C. glutamicum had 84.0% amino acids relative to the standard value and was similar to that of the control. The results of this study suggest the possibility to reduce the toxicity of a grave land containing animals with this disease.

Introduction

Episodes of exotic Newcastle disease in the United States, bovine spongiform encephalopathy in Europe and elsewhere, chronic wasting disease in deer and elk in North America, and foot-and mouth disease (FMD) in the United Kingdom have raised questions about the biosecure disposal of animals with these diseases. The disposal of carcasses is of interest in other situations, from major disease outbreaks among the wildlife to road-kill and injured-animal cases [3].

FMD is a highly infectious and viral disease affecting the cloven-footed animals and is quickly spread by the direct and indirect contact with contaminated individuals as well as through the air [15]. The by-products produced from the decay of carcasses with FMD are an important problem because of the risk of environmental pollution, especially soil and groundwater pollution. Therefore, it is necessary to find the best way to prevent spreading contaminants in humans and other animals.

Microorganisms considerably alter the characteristics of the ecosystem in which they live by causing chemical changes in their metabolic activities. Although the past few decades have focused on describing metabolic diversity, very little is known about their metabolic activity levels in nature and mechanisms on the ecosystems [7].

Therefore, the purpose of this study was to investigate the effect of Corynebacterium glutamicum on the soil environment with carcasses. This study determined environmental parameters such as temperature and pH, the decomposition course of carcasses, the composition of amino acids in the soil around the carcasses, and root elongation for the measurement of soil conditions.

 

Materials and Methods

Microorganisms and Growth Condition

Hansenula polymorpha ATCC CBS4732 and Corynebacterium glutamicum ATCC 13032 (America Type Culture Collection, VA, USA) were used in this study and grown in YPD and Luria–Bertani media [8] at 37℃, 160 rpm and in Luria-Bertani medium at 30℃, 150 rpm, respectively. C. glutamicum was prepared by inoculating in Luria–Bertani medium [13], and the inoculated cells were grown to an optical density (OD) of 4.11 at 30℃ for 20 h with constant stirring velocity (150 rpm). Then, the microorganisms were collected after centrifugation at 15,000 rpm for 20 min and suspended in 1% yeast extract [5] and re-centrifuged. The collected cells were transferred to 0.5 L of a medium containing 1% yeast extract. The dry cell mass density (g/l) was 6.69 g/l.

Carcass

Swine carcasses (~10 kg weight) aged 15–21 days were used as the model of the animals with FMD. Swine was injected with the virus and killed by electric shock immediately prior to the burial.

Experimental Design and Soil Sampling

A total of two carcasses were used in the burial trial. Each carcass was placed in an experimental reactor with a volume, width, height, and diameter of 50 L, 52.5 cm, 44.5 cm, and 39.5 cm, respectively, at a depth of ~15 cm from the soil surface to the base of the upper soil. Moreover, a part of soil was mixed with the microorganism and a medium containing 1% yeast extract. A sequential observation was conducted following 5, 12, 19, and 26 days of burial. The control without microorganism was included with lime and sampled concurrently. After a month, soil samples were collected from each reactor (including the control burial). Soil samples were collected from around the carcass (~10 cm apart). All the soil samples were stored at 4℃ until analysis.

Amino Acid Analysis

The composition of the amino acids in the soil samples was measured by high-performance liquid chromatography (HPLC; Waters Alliance 2690 Analytical HPLC system) equipped with a Nova-PakTM C14 column and a Waters 747 scanning fluorescence detector (Waters Co., USA).

Root Elongation Test

The root elongation toxicity test was conducted according to US EPA protocols, as modified by Chang et al. [5]. The experiments were tested in triplicates in the same petri dishes (60π) on soil directly. The soil samples were serially diluted to different concentrations ( 0%, 50%, a nd 1 00%) and m ixed w ith sea sand (20 meshes). One species of a monocotyledon, oats (Avena sativa), used in this study (using seeds) was purchased from a local supplier, and the seeds were stored at room temperature until used in the tests. For each dish, 10 seeds were placed on the top of the test soil mixture. All the dishes were incubated at room temperature for 5 days, and the temperature was varied from 20℃ to 25℃. At the end of the 5th day, the total length of root was measured using a vernier caliper.

 

Results

Environmental Parameters

In this study, the parameter depended on the outside conditions of the reactor. Fig. 1A shows that at ambient temperature for a month, the maximum and minimum decompositions were recorded at 32℃ and 25℃. The average temperature was 28.2℃ during a month in the season of the summer/fall. Moreover, no change was observed in the pH of the soils collected from near the carcass (pH 7.9) (Fig. 1B). The conditions were suitable for the activity of C. glutamicum.

Fig. 1.Temperature (A) and pH values (B) of the soil samples.

Decomposition of the Carcass

Putrefaction was observed in the buried carcass during a month of burial. By day 5, the buried carcass had started to decompose from a part of the head. Moreover, the soil near the carcass was discolored by the decomposition of the carcass. By day 12, the decomposition was characterized by the beginning of the release of the carcass fluids into the soil. By day 19, the soil level above the carcass had caved partially, because a part of the body had deflated by putrefaction/liquefaction. By day 30, decomposition of the carcass increased and the upper soil caved generally. Upon exhumation after a month, the carcass was decomposed by the degradation of skin and guts and skeletonization. Moreover, leachate was generated by the release of the decomposition fluids from the buried carcass (Fig. 2).

Fig. 2.Time profiles of the decomposition of a carcass for 1 month. After a month of burial, the reactor with the buried animal was dismantled and the carcass decomposition condition was confirmed.

Viability of Microorganisms

Confirming whether microorganisms could survive in the environment contaminated with wastewater generated from the decomposition of carcasses was necessary. C. glutamicum among the two microorganisms showed a survival rate of 28.8%, which was higher than that of the control at the 20% wastewater (Fig. 3A). However, Hansenula polymorpha only had a survival rate of 21.2%. In this study, we examined the compositions of amino acids in the burial soil contaminated with the by-products from carcass decay to confirm whether amino acids increased after treatment with C. glutamicum after a month of decomposition. As shown in Table 1, the contents of amino acids in soil treated with C. glutamicum were generally higher than those of the amino acids in the soil treated with lime.

Fig. 3.Growth of C. glutamicum (A) and H. polymorpha (B) in fluid from the decomposition of the carcass in soil.

Table 1.aSoil sample treated with lime and no C. glutamicum. bConcentrations of amino acids in the presence of C. glutamicum-treated soils; excepted amount of amino acids in medium containing 1% yeast extract.

Root Elongation

Root elongation values of oat were measured 5 days after the planting (Table 2). The length of the oat root showed less toxicity in the soil samples treated with C. glutamicum than those of the untreated samples. The root length in the untreated samples was elongated by 28.1%; however, the oat root in the samples treated with C. glutamicum was similar to that of the control (84.0% relative value) (Fig. 4), probably because the increased composition of amino acids in the soil by C. glutamicum treatment strengthened the plant root and eliminated the toxicity of the soil contaminated from the carcass decomposition.

Table 2.aSeeds were planted in the burial soil samples after 5 days of incubation at room temperature. All the results represent the data from at least three independent experiments. bRoot length was measured 5 days after planting. cSeeds were planted in the soil untreated with C. glutamicum, but with lime. dSeeds were planted in the soil treated with C. glutamicum, but no lime.

Fig. 4.A. sativa (oat) root length in soils untreated (A) and treated (B) with C. glutamicum, 5 days after planting.

 

Discussion

Animal carcasses must be handled properly to prevent hazard to people, water, and the overall environment. Disease can be spread to people and animals if dead livestock is disposed of improperly. Groundwater is also more likely to become contaminated by leachate [12]. In this study, based on the metabolic ability of C. glutamicum, this microorganism was applied for the disposal of livestock carcass burials. The effect of temperature on the soil respiration, microbial biomass, and enzyme activities indicated that the soil microbial biomass plays an important role in the decomposition of carcasses [9]. Moreover, it is important to note that the decomposition progress of a carcass is commonly attributed to the temperature variation [4]. The average temperature in the reactor was 28.2℃ during a month and no change was observed in the pH of the soils. These results show that the conditions were suitable for the activity of C. glutamicum. In addition, this study describes the process of decomposition of a carcass. Decomposition can begin almost directly after the death and progresses through four general stages: autolysis, putrefaction, liquefaction, and skeletonization [4]. From Fig. 2, the carcass treated with C. glutamicum was decomposed by the degradation of skin and guts and by skeletonization. The skeletonization was characterized by the loss of muscle tissue and exposure of the skeletons [1,2]. Carcasses of a small mass (~20 kg) decomposed more rapidly than larger carcasses (~50 kg). Moreover, carcass decomposition followed a sigmoidal curve where approximately 80% of carcass mass was lost [14]. C. glutamicum-treated carcass decomposed significantly faster during 30 days of burial, due to the positive effect by C. glutamicum that showed a survival rate of 28.8%, which was higher than H. polymorpha. Moreover, the percentage of glutamate, proline, threonine, and histidine was enhanced by environmental pollution by phenol in C. glutamicum [10,11]. The contents of amino acids in C. glutamicum-treated soil were generally higher than those of the amino acids in the lime-treated soil (Table 1). The data provide the possibility of the application on actual contaminated soil environments. This study shows that the toxicity of contaminated grave land soils with diseased animals can be decreased. The composition of amino acids in the C. glutamicum-treated soil was generally higher than those of the amino acids in the untreated soil. Some plant species are suitable for phytoremediation. There is evidence that grass species such as corn and barley have varieties that display significant heavy metal tolerance [6]. Root elongation using C. glutamicum-treated soil was conducted to confirm the toxicity of the soil on oat (Avena sativa). The plant root length in the soil treated with C. glutamicum was similar to the soil of clear condition.