Isolation and identification of thermophilic cellulase producing bacteria and its potential application in bioconversion of waste paper

Cellulose, an important plant polymer, is the most abundant and renewable carbon source on earth. Cellulases are inducible enzymes produced by microorganisms to degrade cellulose when cellulosic materials are supplied as substrates. Application of cellulase enzymes in paper, detergent and leather industries demands the finding of highly stable enzymes active at extreme pH and temperature. The search for enzymes from extremophilic microorganisms is one of the ways to obtain enzymes with suitable characteristic features for industrial applications. Practically there are some advantages in using thermostable enzymes in industrial processes as compared to thermolabile enzymes. The main advantage is the increase in rate of reaction as the temperature of the process is increased. At high temperatures the solubility of many polymeric substances is considerably increased and the risk of contamination is reduced. Eventhough a large number of microorganisms are reported for cellulose degradation, only a few of these produce significant quantities of cell-free enzymes to degrade crystalline cellulose in vitro. Till date very few thermophilic cellulolytic bacteria have been described for its cellulase activity. Thermophilic enzymes play a significant role in bioremediation because higher temperatures applied during waste treatment favours the solubility of the substrates. Hence thermophiles remain an important source of cellulolytic enzymes. The arid environment in Oman is very unique in terms of its biodiversity. The probability of getting microbes with distinctive features is more in such environment. The hot springs in arid region like Oman would be a potential source of microbes with novel features. Till date the hot springs in Oman are unstudied resources in terms of its bacterial diversity. Our preliminary studies showed the presence of thermo, acidic and alkaline stable cellulase producing thermophilic and hyperthermophilic bacteria in the Al Kasfah Hot Spring, Al Rustaq, Oman. The increase of solid wastes is becoming a global problem. Different methods such as burial, incineration and recycling are used to dispose solid wastes. Improper management of solid wastes contaminates air, soil and water. In addition, disposal of solid wastes in landfills pollute the ground water and cause emission of greenhouse gases such as carbon dioxide and methane which inturn cause climatic changes. Municipal solid waste contains high amounts of cellulose, which is an ideal organic waste for the growth of most of microorganisms. Municipal solid waste is composed of 40 50% cellulose, 9 12% hemicelluloses, and 10 15% lignin on a dry weight basis. Most of the carbon dioxide and methane are produced from biodegradable cellulosic wastes such as wood, leaves, other agriculture residues and waste papers. Hence, recycling of such cellulosic wastes would decrease the greenhouse effect. Today, environmental policies and regulation progress lead to the development of biodegradation processes to turn organic wastes into valuable products using potential microbes. One of the strategies of Oman government is to findout the possibilities of utilizing alternative renewable resources for energy production. Cellulosic wastes such as paper, wood, agriculture residues and cardboard could be considered as one of the main renewable energy sources. According to 210 survey, about 1.6 million tons of solid waste is simply dumped in landfills in Oman every year. Out of which, 62 thousand tons is of paper waste. This much of paper waste could be used as a source to produce an economically important cellulase enzyme. In addition, using the cellulase enzyme the paper wastes could be degraded and the degraded product could also be used as a substrate to produce commercially important products. So far, there are no national projects that deal with the bioconversion of wastes. Hence the present project would be conducted with an objective to isolate cellulase producing thermophilic bacteria from the hot springs present in South Al Batinah and Muscat regions of Oman. The selected cellulase potential strains would be used to utilize paper wastes. This results in the formation of glucose which could be used to produce value-added products. This study will be conducted in three phases. In the first phase cellulase producing thermophilic bacteria will be isolated from hot springs and identified using 16SrRNA/Ion Torrent sequencing. The identified microbes will be lyophilized and maintained as a thermophilic cellulase producing bacterial culture collection in the department of Biology, Sultan Qaboos University, Oman. In phase 2, some efficient cellulolytic bacteria would be selected to do cellulase fermentation in a laboratory scale fermentor. Increased cellulase production would be achieved by a gradient feed of substrate and maintenance of process conditions at their optimal. The produced enzymes would be purified by ammonium sulfate precipitation, dialysis, gel filtration chromatography. The purified enzyme would be characterized by studying the activity and stability of cellulase at different temperature, pH, salinity, different metals, detergents etc. Molecular weight and the amino acid sequence of cellulase would be characterized using MALDI TOF TOF. In the third phase the characterized bacteria or consortium of bacteria would be used to convert the paper waste to glucose in a laboratory scale fermentor. With the help of suitable microorganisms the glucose would be used to produce biofuel and bioplastic. This proposed research program is an unique and challenging attempt to solve the problem of increasing accumulation of solid waste which could cause environmental and public health concerns in the country. In addition, this research will give knowledge about the bacterial biodiversity in the major hot springs of Oman, helps in capacity building and establish a bioprocess research facility at Sultan Qaboos University.