Based on the study, it shows that biological devulcanization has a high potential as an environmental friendly way for devulcanization of waste rubber in the future. Therefore, the objectives of study for this project are:
• To screen the wavelength for optical density study and parameters for production of tetrathionate hydrolase enzyme from Thiobacillus ferrooxidans.
• To optimize the production of tetrathionate hydrolase enzyme from Thiobacillus ferrooxidans in a stirred tank bioreactor
• To optimize the devulcanization process condition using the optimized tetrathionate hydrolase.
In this research, Thiobacillus ferrooxidans was propagated based on previous study (Ainie& Faridah, 2011). For production of tetrathionate hydrolase the parameters involved were screened using Plackett Burman and One-Factor-At-a Time (OFAT) in shake flask. The selected parameters were further optimized in stirred tank bioreactor using Design Expert® version 6.0.8 software Face Centered Central Composite Design (FCCCD). Then, the enzyme accumulated from the optimized condition was used for the devulcanization process where the process condition involved was studied using OFAT and optimized using FCCCD of Design Expert.
1.6 SCOPE OF RESEARCH
Commonly, devulcanization was done by introducing the rubber crumb during the cultivation of the bacterium. In this research, the tetrathionate hydrolase enzyme produced from Thiobacillus ferrooxidans was extracted first after cultivation process and the enzyme was directly exposed to the rubber surface.
The bacterium Thiobacillus ferrooxidans was first propagated using media and condition based on previous study (Ainie& Faridah, 2011). Then, to maximize the production of the enzyme the parameters such as agitation, inoculums size, temperature, aeration and also five of the cultivation media component (potassium dihydrogen phosphate (KH2PO4), ammonium sulfate ((NH4)2SO4), magnesium sulphate heptahydrate (MgSO4.7H2O), calcium chloride dehydrate (CaCl2.2H2O), and sodium thiosulfate pentahydrate (Na2S2O3.5H2O)) was screened in shake flask using Plackett Burman before it is studied using OFAT. The enzyme produced was analyzed for enzyme activity and total protein every day starting from the initial day. The growth of the bacterium was also studied through optical density. The wavelength used to study the growth was screened to obtain the optimum wavelength to be used throughout the study.
After screening and OFAT study, the parameters were further optimized using design of experiment (DOE) in Design Expert® version 6.0.8 software in shake flask. The level of parameters involved for optimization using FCCCD was determined based on the OFAT result. The enzyme produced was also analyzed for enzyme activity and total protein every day starting from the initial day. The optimization in bioreactor was done for aeration and dissolved oxygen while maintaining other physical parameters as constant based on the optimization result in shake flask.
The enzyme produced by optimized conditions was then used for the devulcanization of rubber tire. For this step, the parameters involved such as time of incubation, enzyme to rubber ratio, and enzyme concentration was studied using OFAT before it is optimized. The experimental design was modeled using DOE in Design Expert® version 6.0.8 software FCCCD. By using these values, it is expected to give optimum conditions for devulcanization process.
1.7 SIGNIFICANT OF STUDY
Compared with the other method of devulcanization, enzymatic devulcanization has many advantages in term of cost, renewable and regenerating. According to Ramirez et al. (2004), tetrathionate hydrolase is proposed to be the enzyme that degrades the sulfur crosslink in the devulcanization process as it catalyzes the metabolism of tetrathionate to sulfate. Then, by applying this method, it can minimize the waste rubber product and directly minimize the pollution because this method is environmentally friendly.
At present, few numbers of biotechnological processes exist for the reuse of waste rubber material. Although a number of patents exist for the biotechnological desulfurization of rubber material exist, but for now none of the processes is currently in commercial (Bredberg et al., 2005). This research was dealing with the optimization of process condition in stirred tank bioreactor for the production of tetrathionate hydrolase from Thiobacillus ferrooxidans and usage of the enzyme directly for devulcanization of the rubber tire. The method applied is hoped to improve the devulcanization rate so that it can be used commercially.