Enhancing the Conversion of Organic Waste into Biofertilizer with Thermophilic Bacteria

Introduction

Composting is a method commonly used to transform biodegradable waste into useful soil amendment products. The composting of organic wastes is not only an ecologically friendly waste disposal system, but it also provides compost to maintain soil productivity and a sustainable agriculture (Narayan, 1994). The process of composting can be described as the accelerated degradation of heterogeneous organic matter by a mixed microbial population in a moist, warm, aerobic environment under controlled conditions. Compost maturity is an important parameter of compost quality (Bernal et al., 2009), and the inoculation of thermophilic microbes has been suggested as a potential approach for accelerating the composting process (Tsai et al., 2007).

Composting is very popular in Taiwan and Asian countries as a means of recycling agricultural by-products and poultry, livestock, industrial, and municipal wastes (Lin et al., 2011b). Such wastes are commonly mixed with various biological materials, and the resulting organic mixture is then inoculated with the appropriate microorganisms, with the production of biofertilizers as the end product. These biofertilizers can then be used not only to return nutrients directly to the soil but also for a multitude of other purposes (Chang and Yang, 2009).Various commercial wastes can be composted. These materials include condensed molasses fermentation solubles (CMS) (Chien and Chen, 2007) and rice bran (Ludwig and Tackett, 1991). Sawdust is usually used as a bulking agent for moisture adjustment in the compost (Hanajima et al., 2006).

Although a variety of microorganisms survive during the composting process, most of those used to date have been mesophiles that can only be used under mesophilic conditions. These types of microorganisms are not appropriate for biofertilizer preparation at higher temperatures (over 50°C). Some studies have demonstrated the efficacy of thermophiles inoculation on compost (Nakasaki et al., 1996; Sundberg et al., 2004; Tang et al., 2004; Zhao and Wong, 2010). Given that some composting materials, such as rice bran, maintain a high temperature in a compost pile when mixed with other materials for composting (Khyami-Horani, 1996), the inoculation of such compost piles with thermophilic bacteria would appear to be a feasible strategy (Chang and Yang, 2009).

In the study reported here, we isolated thermophilic bacteria from the composting process and investigated the effect of these bacteria on the maturity and quality of compost as biofertilizer. The germination of Chinese cabbage (Brassica paruchinensis) seeds in various combinations of compost-amended peat was subsequently assessed to examine the usability of the compost.

Materials and Methods

Results and Discussion

Three thermophilic strains were isolated from the composting aquaculture wastes. These were identified in a BLAST comparison in the NCBI database, with 16S rRNA similarity in the range 97.5%–99.2%, as Bacillus licheniformis, Gordonia terrae, and Virgibacillus halophilus. The phylogenetic tree analysis of these thermophilic isolates (Fig. 1) clearly illustrates that isolates 1, 2, and 3 are closely related to the species B. licheniformis (GU967449), G. terrae (EU590659), and V. halophilus (AB243853), respectively. The morphological and physiological characteristics of the isolates were analyzed and are given in Table 1. B. licheniformis is Gram-positive and a motile rod that grows under both aerobic and anaerobic conditions at temperatures ranging from 35°C to 70°C. In our study, B. licheniformis showed significantly high growth rates (1.73 h⁻¹) and cellulase, catalase, and oxidase activities. These characteristics of the isolated B. licheniformis are similar to those of the B. licheniformis examined by Khyami-Horani and Ryckeboer (Khyami-Horani, 1996; Ryckeboer et al., 2003). G. terrae is a Gram-positive, nonmotile bacteria that can grow only under aerobic conditions at temperatures ranging from 25°C to 65°C. It possesses cellulase activity (142 units/L). G. terrae has been found in hot synthetic compost (Dees and Ghiorse, 2001). Diogo et al. (2010) revealed that the genus Gordonia possesses nitrogenase reductase and suggested that Gordonia is involved in the nitrogen metabolic reaction of compost. V. halophilus is Gram-positive and a motile rod that is unable to survive under anaerobic conditions. This microbial species can grow at temperatures of 30°C–65°C and has cellulase, catalase, and oxidase activities. V. halophilus can grow in both the absence of NaCl and the presence of 18% NaCl (An et al., 2007), and the maximum specific growth rate (0.85 h⁻¹) is lower than that of B. licheniformis and G. terrae. In general, Virgibacillus sp. is positive for nitrate reduction and glucose oxidation, but negative for glucose fermentation (Chen et al., 2009). The Virgibacillus might involve in the energy process of glucose oxidation utilized in biochemical processes in the compost based on its physiological characteristics and composting materials (CMS). Since the V. halophilus possesses cellulase activity, the mechanism for enzymatic cellulose hydrolysis functioned by Virgibacillus should involve synergistic actions by endoglucanase, exoglucanase, and β-glucosidase (Zhang et al., 2006).

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FIG. 1.

Neighbor-joining phylogenetic tree, based on 16S rDNA sequences, showing the positions of isolates 1, 2, 3, and related taxa. Numbers at nodes are the percentages of occurrence in 1000 bootstrapped trees; only values greater than 70% are shown. Bar, 0.1 substitutions per nucleotide position. The outgroup strain used to root the tree was Staphylococcus aureus (HQ260332).

Most importantly, all three bacterial strains isolated showed cellulase activity (142–432 units/L), which favors the degradation of sawdust and rice bran. To date, there have been no reports of G. terrae and V. halophilus possessing cellulase activity. Since cellulose in compost is difficult to decompose rapidly, our results indicate that the composting process could be accelerated through the addition of these cellulose-degradable bacteria to the compost material. After inoculation of the sterile compost pile with B. licheniformis, G. terrae, and V. halophilus, the compost temperatures increased rapidly during the composting process, beginning at room temperature, peaking on day 7 (65°C), leveling off for 3 days, and then decreasing gradually (Fig. 2). It is generally accepted that when the temperature in compost decreases, the compost has reached the stable stage. In comparison, the temperature of the uninoculated compost slowly increased to 62°C on day 21. When the sterile compost, inoculated with three isolated thermophilic bacterial strains, has reached the maturity stage (the 18th day), the bacterial composition of the sterile compost included the mesophilic bacteria (5×10⁵ cells/g-dry raw material), B. licheniformis (2×10⁶ cells/g-dry raw material), G. terrae (8×10⁶ cells/g-dry raw material), and V. halophilus (2×10⁷ cells/g-dry raw material). To confirm the activity or survival of these isolated strains in the compost, we determined their cell numbers on day 7. Results indicate that their population densities in the compost ranged from 3×10⁹ to 9×10¹⁰ cells/g-dry raw material, and their species were re-checked by molecular techniques. Apparently, the composting period was shortened by approximately one third when the three strains of thermophilic bacteria were inoculated in the compost. Similar results have been reported in other studies (Nakasaki et al., 1996; Chang and Yang, 2009). The most likely explanation of our results is that these inoculating bacteria can survive in a wide range of temperatures and have varied oxidation activity in the compost.

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FIG. 2.

Temperature profiles during the composting of aquaculture wastes inoculated or uninoculated with the three isolated thermophilic bacteria. Results are means of triplicate experiments (SD is indicated with error bars).

Germination assays with Chinese cabbage seeds was performed to evaluate the compost quality. The highest germination percentages were obtained in the 25% compost/75% peat mixture (30 g peat:10 g compost), namely, 85%, 92%, and 99% germination after 5, 10, and 15 days of cultivation, respectively (Table 2). Conversely, the highest percentage for the uninoculated compost product was 83% germination after 15 days of cultivation. Ostos et al. (2008) suggested that there was an optimal mixed ratio of compost and peat with optimal nutrient ingredients that would favor initial radicle growth and that, conversely, a high concentration of compost or peat would be unfavorable to radicle growth due to the environmental condition of high osmotic pressure or poor nutrients. The study of Tiquia and Tam used Chinese cabbage seeds to examine the compost maturity of a spent pig litter/sludge mixture and found that the optimal germination percentage was 88% (Tiquia and Tam, 2000). These results suggest that the biofertilizer produced by fast composting is a potentially beneficial and useful material.

Thermophilic bacteria with cellulase activities were isolated from composting material consisting of CMS, rice bran, and sawdust. The inoculation of these composting aquaculture wastes with the three isolated thermophilic strains accelerated the degradation of these organic materials and successfully converted them into biofertilizers. Thus, fast composting through the inoculation of the appropriate bacteria has the potential to shorten the composting period. The quality of the compost obtained by our fast composting process was assessed to be satisfactory based on the results of germination assays with Chinese cabbage seeds.

In this study, we have successfully demonstrated the advantageous utilization of thermophiles to enhance the conversion of waste into biofertilizer. A previous study of similar innovative treatment technology, proposed by Lin et al. (2011a) for the treatment of diesel contaminated soil, indicated that the presence of thermophiles in composting material should have a potential to treat petroleum-related contaminants utilizing food waste thermophilic composting processes. Both studies have demonstrated the added benefits of utilizing thermophiles. These findings were significant, because most previous studies for biological treatment were carried out under mesophilic conditions, but a few were carried out under thermophilic conditions. Future research in the application of thermophiles are warranted and may open up promising scientific contributions.