|Nur Zaida Zahari||Faculty of Science and Natural Resources Universiti Malaysia Sabah, UMS Road, 88400 Kota Kinabalu, Malaysia|
|Piakong Mohd Tuah||Bioreg Technology Solutions Sdn. Bhd., Block H, Lot 3, 2nd Floor, Kian Yap Road, Inanam Capital, 88450 Kota Kinabalu, Malaysia|
|Nur Hazwani Che Zulkifli||Faculty of Science and Natural Resources Universiti Malaysia Sabah, UMS Road, 88400 Kota Kinabalu, Malaysia|
|Fera Nony Cleophas||Faculty of Science and Natural Resources Universiti Malaysia Sabah, UMS Road, 88400 Kota Kinabalu, Malaysia|
Composting is a natural process that occurs as a result of microbial succession and marks the degradation and stabilization of organic matter in waste. The use of microbial additives during composting is believed to be highly efficient, as it increases the production of various enzymes, resulting in a faster rate of waste degradation. The process converts waste into a humus-like substance that can enhance the physical, chemical, and biological properties of soil. In this study, to assess the contribution of microorganisms to the composting process, we selected the thermophilic bacteria degrading Bacillus subtilis-ReK1Hs-Cr1 as the inoculant. The analysis of temperature, pH, moisture, microbial population, compost maturity, seed germination test, and C:N ratio were all monitored during the composting process. Physical changes in the compost, such as color, texture, and smell, were also observed. The findings showed that the inoculation of this thermophile with the addition of chicken manure was able to raise the temperature up to 67.3oC, which may have resulted in the elimination of pathogens, which is above the recommended temperature level of 55oC for 3 consecutive days. There was an increase in the microbial population (4.6 x105 -2.9 x107) at the start of the experimental works and the temperature decreased as the composting process proceeded because of the maturity phase. The pH; 7–9 and moisture content; 51–63 % of compost materials were in the range of optimum composting conditions. The weight of the compost materials in all three treatments, except for the control, decreased by 47% to 55%. The color of the compost turned to dark brown, the obnoxious odor was eliminated, and the texture changed to lose and easy pulverize at the final stage of composting. High germination index (GI) was obtained from the test, indicating that the final product is free from phytotoxic substances and thus suitable as a soil conditioner/fertilizer. Therefore, B. subtilis ReK1Hs-Cr1 is the best candidate as an inoculant added with chicken manure to degrade OPEFB on a large scale using windrow technology.
Bacillus subtilis-ReK1Hs-Cr1; Composting; Germination index; Oil palm empty fruit bunch; Thermophilic strain
Oil palm production is a major agricultural industry in Malaysia. As a result, there has been an increase in the amount of waste generated by the oil palm industry, especially empty fruit bunches (EFB). Solid wastes from oil palm plantations, such as trunks, fronds, and oil palm empty fruit bunches (OEEFB), are a source of concern for the environment. As a result, composting is preferred and environmentally sound, whereby organic waste is reduced to organic fertilizer and soil conditioners through biological processes (Shukor et al., 2018; Pan, Dam, and Sen, 2012). In composting, the higher-plant material breaks down under the influence of aerobic thermophilic microorganisms present in the waste to a material rich in organic nutrients. Oil Palm Empty Fruit Bunch (OPEFB) fiber is a lignocellulosic waste from palm oil mills that contains 12% lignin and 75% cellulose (Hermansyah et al., 2019). Lignocellulose components are the main building block of plant cell walls where the cellulose fibrils are embedded in an amorphous matrix of lignin and hemicellulose (Zoghlami and Paes, 2019). Lignin is a structural polymer found in vascular plants, which plays essential functions in their survival. It provides rigidity to the plant and binds plant cells together, imparting resistance to impact, bending, and compression. However, cellulose is a highly regular polymer of any hydro-D-glucopyranase unit linked with ß (1-4) linkage, and this molecule can be easily metabolized by various cellulolytic microbes. Bacteria play a significant role in the humification processes associated with soil and compost. The enzymatic ability to cleave alkyl-aryl ether bonds enables bacteria to degrade oligomeric and monomeric aromatic compounds released during lignocellulose degradation. Composting has been used as a system for the disposal of organic waste, to produce organic fertilizer, and to improve soil biological fertility. Zulkepli and Tuah (2008) reported thermophilic bacteria had the potential to enhance the biocomposting of OPEFB which can reduce the weight of OPEFB and cause lignocellulose loss in 7 days of degradation. He also reported that the maximum temperature achieved during the composting of OPEFB by lignocellulose-degrading bacteria was only 45oC which did not meet the minimum requirement of 55oC for three consecutive days sanitized compost material as suggested by Jones and Martin (2003). The four most commonly used composting methods in the industry are open static piles, turning windrows and piles, aerated static piles, and in-vessel systems. Among these options, an in-vessel composting system has many advantages because it requires less space and provides better control over agitation, aeration, and mixing of the compost materials. Typically, two phases are engaged in in-vessel composting systems: a high-rate phase in the vessel composter and a curing phase in an outside composting pile. In this study, a locally isolated thermophilic lignocellulose-degrading bacteria from a hot spring (Bacillus subtilis ReK1Hs-Cr1) (Zulkepli and Tuah, 2008) were investigated for its capability to enhance composting of shredded OPEFB by using an in-vessel composting system. Non-inoculant and inoculant bin-type composting of OPEFB were compared by monitoring parameters which include temperature, microbial population, pH, C: N ratio, and Germination Index during the 90-day period.
2.1. Organism maintenance and preparation
Bacteria used in this work was the lignocellulose-degrading thermophilic strain Bacillus subtilis-ReK1Hs-Cr1 obtained from a hot spring in Ranau, Sabah (Zulkepli and Tuah, 2008). This strain has been selected due to its capability to sustain high temperatures, thus helping in elevating the temperature of compost materials and ensuring the elimination of both animal and plant pathogens during the composting process. The organism was maintained on Nutrient Agar (2 g/L yeast extract, 2 g/L meat extract, 5 g/L peptone, 4 g/L NaCl, 10 g/L glucose, and 12 g/L agar in a liter) at 4°C (Zahari et al., 2021a). Subcultures were made weekly and cultivated at 40°C. To achieve successful and rapid composting of shredded OPEFB, inoculation of bacteria was needed. The sterilized raw materials were inoculated with 10% broth (Zahari et al., 2022) inoculum (107 CFU/mL) of Bacillus subtilis-ReK1Hs-Cr1.
2.2. Composting process
Lab-scale in-vessel composting was carried out in accordance with Figure 1. Black bins with a capacity of 69 L and a height of 0.65 m (65 cm) were prepared. Approximately 10 holes with diameters of 1.5 to 2.0 cm were drilled at the bottom of each bin to collect leachate and similar holes were drilled on the sides of the bins for aeration purposes. The composting process involved four treatments, each with two duplicates, as shown in Table 1.
Table 1 Various treatments
carried out in this study.
Figure 1 In-vessel composting (a) (bin-type) and (b) arrangement of EFB and chicken manure in the bin.
2.3. Sampling and analysis
The reduction weight of the compost along with color
and texture, was observed at the initial and the end of the composting process.
The temperature of the compost was recorded daily at the surface, middle, and
bottom of the layer by using a digital temperature probe. pH value was analyzed
by using a pH meter (1: 10 waters: extract). Moisture content was analyzed by
drying 10 g of the sample at 1200C until a constant weight was
maintaining the moisture content of all treatments, inoculants were added
according to the weight of the compost materials, which is 5 mL for every
kilogram of waste in the compost weekly. For biomass concentration, the
microbial population (CFU/mL) was tested every 15 days. The Spread Plate
Technique was used to obtain the CFU/mL after 24 h at 40°C (Taliesin and John, 2021). The inoculant was added on
a weekly basis. Leachate was collected and added back into the compost material
to control the moisture content. The
carbon and nitrogen contents were analyzed using CNHS/O analyzer 2000 and
Inductively Coupled Plasma (ICPOES, Perkin Elmer, USA). All samples were done in
triplicates to obtain accurate data. The seed germination index (GI) was
carried out to determine compost maturity and stability. 10 g of green bean
seed ore were placed on filter paper soaked in 5 mL of compost extract. The
petri dish was placed in a dark room at room temperature. For control, 1 g of
seed was soaked in distilled water. The experiment was conducted in triplicates
and incubated for 3 days. After incubation, the root length and seed
germination index (GI) was calculated using the equation by Fetouh and Hassan (2014) below:
3.1. Physiochemical changes during the co-composting process
The changes in temperature during the
90-day composting process of different treatments are shown in Figure 2 (a).
There are four stages included in this process which are mesophilic,
thermophilic, cooling, and maturity compost. The duration of each stage depends
on the initial composition of the mixture in a treatment bin. In the mesophilic
phase, a variety of bacteria, actinomycetes, and fungi quickly break down the
C-rich substrates at moderate temperatures, usually between 15 and 40°C. Heat
is produced by this aerobic metabolism. As time passed, the temperature rose
rapidly to more than > 45°C (Thermophilic phase) in less than 15 hours
during the initial stages. The results show that the temperature was increased
in all treatments studied, describing a peak of microbial activity. For
Treatment 1 (R1), (R2), and (R3), the maximum temperature reached was 58.8oC,
56.8oC and 67.3°C on day four, respectively. The thermophilic phase
of Treatment 3 (R3) was maintained for 14 days, while for other treatments, the
thermophilic phase was maintained for only 8 days. While for the control (K),
the thermophilic phase only lasted 4 days, and the maximum temperature only
reached 50.2°C. This shows that the addition of an inoculant of bacterial
strain and chicken manure has a positive effect on the composting, especially
during the thermophilic phase, that help in elevating the temperature of
compost materials during composting process. The high temperature achieved
during the composting process ensured the elimination of both animal and plant
pathogens. Only three to four days at a temperature of 55°C was sufficient for
eliminating the pathogens (Zhao, Ta, and Wang, 2017). Kim et al. (2019) reported
that pathogens can be killed at 55°C. Additionally, almost all pathogens are
eliminated in a few days making the compost product suitable for agricultural
use. Besides, a rise in temperature during composting are the consequence of
the breaking down of the readily available organic matter and nitrogen
compounds by microorganism (Raut et al., 2008). As time
increase, the organic matter becomes more stabilized, thus slower the microbial
activity and decomposition rate. A steady temperature was achieved at the last
stages of composting, indicating that less degradation (cooling phase) process
took place. The final maturing phase is characterized by lower temperatures
below 25°C and reduced oxygen uptake rates of aerobic microorganisms. During this
stage, degradation of the more refractory organic compounds continues, and soil
meso and macrofauna enter.
Figure 2(b) illustrates the weekly pH changes of the composting process for the different treatments over the 90-day period. The initial pH values of all treatments of the composting process were nearly neutral, which were 7.6 for (R1), 7.5 for (R2), 7.5 for (R3), and 7.7 for Control (K). During the composting process, the pH values for all treatments increased until week 3. For Treatment 1, the pH of the compost material gradually increased to 8.7 at week three from the initial pH of 7.5. The pH value at week three was observed to be the highest pH value, 8.7, throughout the composting process. Then, the pH value slightly decreased from week four to week six. This trend has been observed in all treatments. The pH rise was due to the organic acid volatilization and microbial decomposition (mineralization) that release ammonia from the organic nitrogen sources (Umsakul, Dissara, and Srimuang, 2010). pH for (R1) has been decreased to 7.1 at week 11 then the value raised again to 8.8 in the final stage of the composting process. A similar trend was observed in (R2), (R3), and Control (K). At the final stage of the composting process, the results obtained for (R1), (R2), and (R3) and Control (K) were 8.8, 8.1, 8.5, and 8.1 respectively.
Figure 2 Profile of physiochemical elements that use different treatment conditions. (a) Temperature changes during composting. (b) pH changes during different waste substrates. (c) Changes in moisture content during composting
Previous research found that microbial activity increased the likelihood of achieving a suitable pH range of 5.5–9.0, while the composting process is most effective at pH values between 6.5 and 8.0 (Pan, Dam, and Sen, 2012). The pH of all treatments was alkaline and stable due to ammonia generation, which could improve the composting process by inhibiting pathogenic fungi that prefer acidic conditions (Saidi et al., 2008).
Figure 2 (c) shows the weekly moisture content of
the composting process of different treatments during the 90-day period.
Moisture appears to be a significant component controlling microbial activity (Anastasi, Varese, and Marchisio, 2005), as low moisture content hinders the growth of
helpful microbes (Tiquia, 2010), whereas excessive moisture might cause anaerobic conditions, resulting
in the creation of unpleasant odors and poisonous volatile compounds (Saidi et al., 2008). The
moisture content of Treatment 1 (R1), Treatment 2 (R2), Treatment 3 (R3), and
Control (K) was shown as 51 %, 56 %, 52 %, and 55 %, respectively. The moisture
content for all treatments was increased when the temperature of the compost
was decreased. This high moisture level in all treatments resulted in a cooling
effect. This is due to the gaseous exchange by reducing diffusion and
consequently constraining oxygen uptake by microbes, thus resulting in lower
microbial activity (Zahari et al., 2021b). The average moisture content for Treatment 1 (R1)
was 59.5 % throughout the composting process. The moisture content was
maintained in the range of 52 % to 62 % for the Control (K) throughout the
composting process. The moisture content for Control (K) decreased from 62 % at
the begining to 55% during the final stage of the composting process. For (R2),
the moisture content obtained in the initial stage was 58 %. The moisture
content slightly increased to 58 % at week 1. The moisture content showed a
gradual decrease at week two to 44 % even though inoculants were added to all
bins as usual and increased again to the optimal range (61%). At week three,
there was a slight increase and decrease in moisture content of (R2) until week
11. At week 11, the moisture content noted an obvious decrease to 48 % and
continued to decrease to 47 % in week 12. The decrease in moisture content may
be caused by the decrease in microbial activity in compost, which has a direct
effect on a high content of organic matter (Kazami et al.,
2016). Generally, the moisture
content for all treatments showed a decreasing trend till the end of the
composting process. The addition of inoculants into the compost materials is
adequate to maintain the moisture content of the treatments. Occasionally,
leachate from the compost was collected and added to the compost materials to
maintain the moisture content. A comparative study in Table 2 showed the
performance of various compost processes using different microbial inoculants
within the present work.
Table 2 Summary of the previous
studies by different microbial inoculants used for composting of EFB
3.2. Bacterial count
The microbial community of different treatments was
studied during the composting process. Bacteria and fungi that constitute the
microbial community structure of the composting environment are present and
active during the composting process. Different bacteria and fungi can have a
beneficial or detrimental impact on the entire composting process. Their
diversity also hints at the composting mechanisms at work. From Figure 3, which
shows the microbial population of different treatments during the 90-day composting
process, the microbial population in the compost material of all treatments was
almost the same for (R1), (R2), (R3), and Control (K), which are 7.2×106 – 2.8×107,
5.0×105 – 2.9×107, 4.6×105 – 2.9×107 and 1.5×106 – 2.9×107 CFUmL-1, respectively. This is because the
compost materials from all treatments were from the same source. The population
of microorganisms is slightly decreased for all treatments from day 1 until day
15 because the high temperature during the thermophilic phase of composting
(> 45°C) killed the pathogens as well as other microorganisms in the
compost. The microbial population, therefore, continued decreasing until the
end of the composting process. This is because the microbial cannot survive
without nutrients and oxygen (Riedel et
al., 2013). While for
the control, the microbial population decreased in week 45, and the compost
materials were in the mesophilic phase. In this phase, it is most suitable for
most of the microorganisms to grow and reproduce. Thus, the population of
microorganisms in the compost increased again.
Figure 3 Profile of microbial population according to the respective composting treatment.
Compost stability and maturity
Figure 4 Germination Index of all treatments of shredded OPEFB composting during the 90-day period.
3.4. Weight reduction of the compost material
The initial weight of the compost materials in all
treatments during the composting process was 76.7 kg, 60.3 kg, 76.6 kg, and
59.8 kg for (R1), (R2), and (R3) and Control, respectively. The weight of the compost material in Treatment 3
showed a significant decrease in weight to 33.3 kg, which is a 57 % reduction
compared to the initial weight. The weight of the compost materials of (R1),
(R2), and Control (K) has been reduced by 54 %, 55%, and 34 %, respectively.
The weight reduction of the compost materials in all treatments was associated
with the evaporation and leaching of moisture (Jain, Daga, and Kalamdhah, 2019).