Biodegradation of leaf compost by thermophilic cellulolytic bacteria isolated from compost

This study aims to isolate and screen cellulose-degrading bacteria at high temperature capacity and to investigate the efficiency of isolated bacteria in breaking down fresh leaf compost. Bacteria were isolated from 4 types of leaf compost consisting of compost Day 7, Month 1, Month 2 and Month 3, where the main raw material for composting was leaf residues collected from Chulalongkorn University. During composting, 4 phases occur: (1) mesophilic (25-40°C), during which easily degradable compounds such as sugar and protein are broken down by microorganisms, leading to a rapid increase in temperature; (2) thermophilic phase (35-65°C), which degradation proceeds rapidly; (3) cooling phase (secondary mesophilic phase); and (4) maturation phase (Abdel-Rahman et al., 2016; Insam and de Bertoldi, 2007; Liu et al., 2011).

Cellulose, an important component of leaf litter (Akhtar, Goyal and Goyal, 2015), is beta-D-glucose chain with a degree of polymerization of 40,000 and links each glucose molecule with -1,4-glycosidic linkage (Insam and de Bertoldi, 2007). For the mentioned reason, this study aims to use leaf compost as a source of effective thermophilic cellulolytic bacteria and to determine the efficiency of the isolated bacteria in degrading fresh leaf compost. Leaf compost samples were collected from the leaf compost piles at Chulalongkorn University and used as a source of bacteria and raw materials for the degradation study.

In the study of leaf compost biodegradation, the parameters used to detect the degradation progress are total organic carbon (TOC), total Kjeldahl nitrogen (TKN) and C/N ratio. Effective thermophilic cellulolytic bacteria can be obtained from leaf compost and have an efficiency to degrade fresh leaf compost.

LITERATURE REVIEW

Composting

Factors affecting composting process
Phases of composting process

Microorganisms in compost .1 Actinobacteria

Bacteria
Fungi

Cellulose
Cellulose degradation by microorganisms

Cellulose degradation by thermophilic microorganisms

Measurement of cellulase activity
Microbial study

Culture medium
Streak plate technique
Morphology study

Related research

In some cases, aeration may be necessary to maintain an adequate oxygen concentration in the compost pile. Temperature is a major factor in the composting process, as it determines the group of microorganisms that break down the organic materials in the compost pile. Proper management is required by watering the compost to keep the moisture content in the optimum range (Richard et al., 2002).

Bacteria are the main group of microbes in the thermophilic phase, due to the diversity of species and large quantities, responsible for the breakdown of cellulose, which is an important component of raw materials for composting. Nitrosomonas europaea has been reported as the domain AOB species in the thermophilic phase of composting. Fungi are most often found in the mesophilic phase (at the beginning and end of composting).

The color intensity depends on the concentration of reducing sugars in the solution, which can be measured with a spectrophotometer 575 nm. Microbes can be classified according to the type of energy source required, such as cellulolytic microbes that can break down carbon in the form of cellulose for use as an energy source. The results have shown that Geobacillus is a domain bacterial species in the thermophilic phase of composting.

In this study, the temperature in the composting process peaked on the second day and was still in the thermophilic phase until the 10th day, in which the elevated temperature is associated with enzymatic activities.

Figure 2.2 The change of temperature during composting process (Sánchez, Ospina, and Montoya, 2017)

MATERIAL AND METHOD

Source of microorganisms
Raw material and source Compost sample

Carboxymethyl cellulose (CMC) broth
Carboxymethyl cellulose (CMC) agar
Luria-Bertani (LB) agar slant

Chemicals and reagents, and special instruments .1 Chemical and reagents 1F 2

Special instrument

Compost sampling and cultivation procedures

Isolation and screening of thermophilic cellulolytic bacteria

Procedures of chemical analysis

Hydrolysis capacity measurement
Determination of cellulase activity

Bacterial morphology study
Degradation efficiency test
RESULTS

Compost sampling

Precision Digital Scale Balance: 40SM-200A, Precisa, Switzerland - Spectrophotometer: 1200, Labomed, inc., United States of America - Vernier Caliper. Ten grams of each compost sample was inoculated into 100.0 mL culture medium and incubated at 60°C under 200 rpm shaking condition for 3 days. Some of each bacterial colony was stored on Luria-Bertani (LB) slants at 4°C for further studies.

Cellulolytic bacteria can use CMC as a carbon and energy source and develop a hydrolytic clearance zone around a bacterial colony. The hydrolysis capacity (HC value) was calculated as the sum of the diameter of the colony and substrate degradation zone divided by the total colony diameter (Awasthi et al., 2018) as shown in equation 3.1. All HC values obtained from all bacterial colonies were statistically analyzed using SPSS software (version 22.0).

A loopful of each selected bacterial isolate was inoculated into 10.0 ml CMC broth and incubated at 60°C, 200 rpm shaking condition for 3 days. The absorbance of the mixture was measured at 575 nm using a spectrophotometer and the reducing sugar production of each bacterium was determined by comparison with the glucose standard curve (preparation was described in Appendix B). Fresh leaf compost (leaf residues mixed with other composting materials) on day 0 of the process was obtained from the same compost pile as the source of bacterial culture using sterile plastic bag and stored at 4ºC until experiments.

Three flasks containing 100.0 g of compost were inoculated with 10.0 ml of bacterial culture as a treatment, while one flask was without microbial inoculum. From the pH measurement using a pH indicator strip, the pH values of all types of compost were similar in the range of 5- the results shown in Table 4.1, the physical properties of each type of compost samples were clearly different D can be observed the texture of. After 3 days of incubation, a colony of cellulose-degrading bacteria appeared on the agar as shown in Figure 4.4 Bacterial colony morphology of all colonies was observed and presented in Table C-1 in Appendix C.

Then, each colony was spotted on fresh CMC agar for a clear zone of cellulose to appear around the colony, as shown in Figure 4.6, and the diameter of the clear zone was measured to calculate hydrolysis. After statistical analysis, the result was displayed as a box plot, as shown in Figure 4.7, the HC value in the third quartile (Q3, there were 11 bacterial colonies with a HC value greater than Q3, as shown in Table 4.2. Colony morphologies of all colonies, obtained by bacterial proliferation, we observed culture solutions on CMC agar, and then we presented the colony morphology of the 11 best selected bacteria in Table 4.3.

Table 4.1 Physical characteristics of compost samples

DISCUSSION & CONCLUSION

Compost sampling
Conclusions
Suggestion

Month 1; Month 2; and Month 3 compost heap, the temperature was measured at the outer zone which has a lower temperature. The temperature of Month 2 is below a thermophilic range because on the day measured, Month 2 compost was watered. Month 3 compost had the lowest temperature at 30.3ºC due to the entry of the ripening phase, which lowered the temperatures.

While compost samples from month 1 and month 2 had a moisture content higher than the appropriate value due to watering. The highest moisture content was found in month 2 compost with a value of 66.4%, according to watering during sample collection. At the end of composting, mature compost should have a moisture content of about 40% (Stentiford, 1996).

Although the moisture content of month 3 compost (ready-to-use compost) was 61.7%, it is not expected to affect compost utilization. At the start of composting, small molecule organics are broken down by the primary decomposer, leading to the production of organic acids, making the pH of the compost slightly acidic (pH around 5). The physical characteristics of the compost have changed with the increasing duration of composting, the size of the raw materials being smaller as time went on.

As the decomposition continued, the texture of month 1 and month 2 became more visible, the size of the leaf remains was smaller. The texture of the 3rd month compost was very similar to loam, and the color and smell were similar to good quality soil. The color of the compost changed from the brown color of leaf litter to the dark brown or black color of humus, which is a stable organic matter obtained through composting.

From Figure 4.2 it could be observed that the texture of the compost sample was obviously changed in Month 1 compost. The texture of the compost and the probability of the highest biodegradation when the time is over for a month were related to HC values. While bacteria strains isolated from Day 7, Month 1 and Month 3 compost samples were and 9.1% percent which respectively obtained HC values greater than Q3.

CONTENTS

CONTENTS (CONT.)

LIST OF TABLES

LIST OF EQUATION

Isolation, screening and characterization of plant growth-promoting bacteria from the rhizosphere of durum wheat to improve the efficiency of N and P nutrients. Additives aided green waste composting: Effects on organic matter decomposition, compost maturity and quality of finished compost. Effect of aeration rate, C/N ratio and moisture content on compost stability and maturity.

A rapid and easy method for the detection of microbial cellulases on agar plates using Gram's iodine. Changes in biochemical and microbiological parameters during the period of rapid composting of dairy manure with rice bran. Dynamics of functional microbial groups during mesophilic composting of agro-industrial wastes and free-living (N2)-fixing bacteria utilization.

Isolation and identification of novel cellulases producing thermophilic bacteria from an Egyptian hot spring and some properties of the crude enzyme. Effectiveness of inoculation with isolated Geobacillus strains in the thermophilic phase of vegetable waste composting.

APPENDICES