Microbial Sciences Division of ARI specializes in solving industrial pollution problems through innovative biological and bio-complimentary technologies. It has rapidly established itself at national and international level in its chosen field with an emphasis on microbial degradation of toxic chemopollutants.

Past Accomplishments

Microbial degradation of nitroaromatic compounds (trinitrotoluene, tetryl and dinitrobenzene) has been studied in detail. Pathway and molecular basis of degradation of dinitrobenzene has been worked out.

Aerobic microbial process has been developed for treatment of phenol bearing dye industry waste. The process was transferred to the dye industry for on-site treatment of the waste using cultures of Pseudomonas alcaligenes and P.mendocina. The process resulted in 100 % removal in phenol and 80% removal in colour of the waste within 48 hours at ambient temperature and is being operated successfully at the factory for last seven years. Biochemical pathway of degradation of the dye methyl violet has been illustrated.

Biodegradation of caprolactam appearing in nylon-6 waste water as a major pollutant has been studied and Pseudomonas putida was found to completely degrade the compound.

A two stage microbial process involving biodegradation 1) under aeration followed by; 2) in fixed film bioreactor has been developed for treatment of Mancozeb pesticide waste effluent at laboratory scale using cultures of Arthrobacter sp. and Microbacterium sp.

Present Scope of Research

Pesticides are important agrochemicals and presently organophosphates are being widely used. However due to their toxicity, their removal from waste waters and contaminated soils becomes necessary.

Studies have been carried out on biodegradation of organophosphorus pesticides namely Monocrotophos and Dimethoate. Bacterial cultures capable of growing on these pesticides have been isolated and identified. Studies have indicated over 90% degradation of both the pesticides by the cultures isolated. Metabolic pathway of degradation of these pesticides has been proposed. Biodegradation of Dimethoate has been found to be plasmid associated. Studies on bioremediation of industrial waste waters containing these pesticides are underway.

Future Projections

Bioremediation of soils contaminated with organochlorine pesticides.

Due to enormous use of organochlorine pesticides in the past and because of their recalcitrant nature, these toxic compounds are accumulated in soils, lake sediments and groundwater.

Since these pesticides enter into food chain through uptake by plants, it has become imperative to remove them for contaminated sites.

Hexachlorocyclohexane (HCH or benzene hexachloride or BHC or Lindane) is a commonly used insecticide and Atrazine, a herbicide. Studies have been initiated on bioremediation of contaminated soils using plant microbe interactive potential. Various plants will be screened and their rhizospheric microflora will be studied for biodegradation of these pesticides. Thus, phytoremediation technique will be developed.

The work is supported by Indo-Swiss Collaboration in Biotechnology (ISCB), Govt. of Switzerland and DBT, Govt. of India.

Biogas Technology

Past Accomplishments

Variety of anaerobic bacteria including hydrolytic, syntrophic and methanogenic involved in biogas production from cattle dung, pig dung and distillery waste have been isolated and identified. Optimum requirements of many of these bacteria have been worked out. Similarly effect of pH, temperature, volatile fatty acids, and salt have been studied for different methanogens. All these studies are useful in avoiding souring and failure of anaerobic digestion of waste water and solid biomass. Preliminary trials on use of some of these cultures as an inoculum to enhance biogas productivity suggest potential in application of such inocula on large scale. Microbiological studies have also resulted in isolating two novel species of bacteria, viz. Methanolobus bombayensis, & Selenomonas lipolytica. The culture collection established at this Institute has over 30 anaerobic bacteria including methanogens and other anaerobes.

On application side of principles of biogas technology, optimum parameters for biogas production from variety of biomass have been worked. These include canteen waste, market waste, piggery waste, banana peels, etc. Some of these have been transferred to industry for its field scale application. The anaerobic treatment technologies have been standardized for application to pharmaceutical waste water, dairy waste, pulp & paper mill waste. Based on this consultancy have been worked out for over six companies.

Modification of conventional single reactor based biogas plant for cattle dung into two stage reactor biogas plant proved very useful for anaerobic digestion of human night soil either exclusively or with cattle dung. Such modification proved essential to inactivate enteric bacterial pathogens likely to occur in human night soil. This finding is very helpful for application of biogas technology in rural area and slum area particularly for clean and safe sanitation. A microbial process termed ARISCOE PROCESS for removal of Hydrogen Sulphide from biogas is developed and transfered to the industry.

Over 10 research projects sponsored by different government agencies like DST, CSIR, ONGC, MNES and private companies have been completed.

Present Scope of Research

Based on laboratory studies 700 L capacity anaerobic digester, a high rate two stage anaerobic treatment method has been developed for distillery waste. The method differs from most of the known treatments since it works efficiently (60-65 % reduction COD) for organic loading rate of 23 Kg COd/M3/day. By this method, distillery waste is digested without any dilution. This makes the process more economical. A novel acid tolerant methanogen has been isolated from first stage digester of this newly developed anaerobic treatment process. This methanogen grows well at pH 6 & grows even at pH 5.0. The isolate on identification proved to be a novel species of Methanobrevibacter and is named as Methanobrevibacter acididurans. As a part of holistic approach for distillery waste treatment, a microbial process to remove hydrogen sulphide from biogas produced during anaerobic treatment is developed. With this process hydrogen sulphide level in biogas could be brought down from 3% v/v to < 0.1 % v/v. The process has 6 min gas residence time. It uses a bacterial culture. It works efficiently at room temperature and able to sustain shock load. The process know-how is transfered to the industry. A patent has been filed for distillery waste treatment method.

Microbially induced corrosion is one major problem faced by offshore oil & gas projects. Through financial grant from Oil & Natural Gas Corporation Ltd. Mumbai, both sulphate reducing and other anaerobic bacteria have been isolated from Mumbai Offshore Oil & Gas project. These have give good insight on microbially induced corrosion and led to develop methods for detection of sulphate reducing bacteria.

The group has established nearly all the facilities and is considered as leading group in the area of anaerobic bacteria and anaerobic treatments and biogas technology. The group has published three books and over 50 papers in this area. The group has also contributed actively in developing manpower to work on anaerobic bacteria and anaerobic treatments. Training course on these aspects were successfully conducted for DST, MNES, ONGC, etc. and are organized from time to time.

Under MNES sponsored project, a low cost microbial technology shall be developed to enhance biogas production from cattle dung is being developed.

A microbial process for removal of color of distillery waste is being developed. Similarly, research on microbial production of Succinic acid is being carried out.

Biodiversity of anaerobic bacteria particularly the methanogens and SRB is being studied.

Future Projections

Developing low cost microbial technology for enhance biogas production from cattle dung at low temperatures.

To develop and commercialize bioinoculum packets for increase efficiency of anaerobic digester, to recover sour digester and to enhance biogas production. Developing a microbial process for degradation of organic halides (AOX) from pulp of paper mill effluent.

To develop rapid check kits for detection of methanogens and sulphate reducing bacteria for its in situ application.

Methanogenesis in rice field.

Production of industrially important biochemicals using microorganisms

Vitamins and amino acids

Past Accomplishments

In the project procedures for production of Vitamin B12 active compounds including Vitamin B12 and Factor III (a substitute for Vitamin B12 in poultry and cattle feed), 5-aminolevulinic acid (a herbicide) and aromatic aminoacids by methanogens as well as development of a foliar spray from proteinaceous wastes and a spore formation procedure for Lactobacillus sporogenes were developed. The foliar spray, Biofert, developed is composed of amino acids and their derivatives, which is useful in obtaining higher yields of crops, fruits, vegetables, etc.

Present Scope of Research

At present the work is mainly carried out on metabolites and enzyme profiles as well as on environment modulations of methanogens. Metabolites labeled with isotopes are in demand for research, diagnostic, medical applications, etc.

Future Projections

Different types of diagnostic kits will be developed for diagnostic implications in hepatitis, liver diseases, myocardial infarction, obstructive jaundice, acute pancreatitis, drug induced hemolytic anaemia, etc.

Enzymes of alkaliphilic and thermophilic bacteria

Enzymes are the active biomolecules catalyzing a number of biochemical reactions in animals, plants and microorganisms. Although plants and animals have been the source of many enzymes, there has been increasing trend to use microorganisms as the source of enzymes mainly because microorganisms grow fast and hence production of the enzymes becomes a quick process; it is easy to handle the microorganisms and recovery is cheap.

Extremophiles e.g. alkaliphilic bacteria produce alkaline enzymes required in certain alkaline reactions (laundry detergents) and thermophilic microorganisms produce thermotolerant / heat stable enzymes useful in certain applications.

At Agharkar Research Institute, the Scientists at Microbial Sciences Division have been actively engaged in exploitation of alkaliphilic bacteria for production of the enzyme protease and thermophilic Streptomyces sp. for production of thrombolytic enzyme having medical applications.

The recent therapy for myocardial infarction is enzyme therapy, which helps to dissolve the fibrin clot and recanalize the occluded blood vessels. Available imported fibrinolytic enzymes are not only costly but sometimes the enzyme fails due to other complications. Due to the significance of fibrinolytic enzyme in the treatment of different types of thrombosis, search for the microbial sources of these enzymes was continued. The present investigation deals with the production of a fibrinolytic enzyme using a thermophilic Streptomyces species, isolated from Lonar crater and hot spring water.

Past Accomplishments

Alkaliphilic bacteria isolated from alkaline lake of Lonar, Maharashtra State were studied for production of the enzymes protease, amylase, lipase and chitinase. Microbial process for production of the enzyme protease using Arthrobacter ramosus and Bacillus alcalophilus and soyacake as the substrate has been developed and worked out to be an economically viable process. The enzyme is stable at alkaline pH (upto 12.0), thermostable (temperature of upto 65^oC) and stable in detergent. It was demonstrated to remove blood stain on cotton fabric and thus can be used as a detergent enzyme.

Thermophilic Streptomyces megasporus strain SD5 could produce more fibrinolytic enzyme than the reported one. The 35 Kda enzyme was proteolytic, fibrinolytic, highly stable at different pH and temperatures. The enzyme could hydrolyse different peptides with a chromogenic tag. The gene encoding the enzyme was successfully cloned in E.coli and the properties of the expressed protein were similar to wild one with more activity.

Present Scope of Research

Scale up studies have been initiated from the point of view of commercialization of the process for production of the enzyme protease. Attempts are being made to increase efficiency of the process by solid state fermentation, mutation of the organism, testing other substrates for production of the enzyme immobilization of the enzyme, etc.

Future Projections

Molecular biological aspects of protease from alkaliphilic bacteria will be studied. Commercialization of process for production of actinokinase will be attempted.

Metal-microbe interactions

We have worked extensively on microorganisms capable of interacting with metals. These studies have provided novel ways of combating environmental pollution caused by heavy metals. This work covers a complete gamut of science ranging from development of laboratory-scale technologies, their scale-up, transfer of technology to industry, consultancy work and fundamental studies on the microorganisms involved. A summary of our work is given below:

Our studies on microbial reduction of metals has led to the development of novel processes for the treatment of waste waters containing highly toxic chromium and remediation of chromium-contaminated soils. The former process is licensed to a reputed environmental engineering company for commercial use. Our discovery that chromium reduction is a plasmid borne property and is catalyzed by an enzyme, chromate reductase has opened up exciting possibilities of creating transgenic organisms, e.g. plants for the control of heavy metal pollution in water and soils. Another spin-off of this work is the possibility of using microbial reduction as a cheaper means of extracting metals from ores.

In an exhaustive study undertaken by us, a large number of microorganisms capable of adsorbing metals were screened and characterized . A new method for pelletizing the microbial biomass was invented and used to obtain a product, viz. metal biosorbent beads. The biosorbent beads technology provides an efficient, metal-specific and cost-effective alternative to the conventional ion-exchange process and can be used for the treatment of effluents from industries such as electroplating, mining, automobile, metal finishing, paint, tanning, jewellery manufacturing, photofilm processing etc. The prospect of using biosorbent beads for gold recovery from jewellery and printed circuit board industry waste water has received wide publicity in India. With minor variations, metal biosorbent technology could also be useful in the remediation of metal-containing solid wastes, soils, metallic particulates, organics, and removal of contaminating metals from pharmaceutical preparations and foods.

The disposal of wastes containing highly toxic cyanide and metal-cyanides is a major problem faced by industries. We have developed a new biodegradation method that takes care of both cyanide and metals and makes treated waste completely safe for discharge. Another ingenious process developed can biorecover metal-cyanides from waste waters for reuse in the industry.

We have also investigated the microbial reactions associated with acid and metal pollution in and around mining areas. This work showed that such a pollution can be contained in a bioleaching operation. As a result, India’s first pilot scale copper bioleaching plant was put up at Malanjkhand Copper Project of Hindustan Copper Ltd. A specialized culture collection and data bank for metal-interacting microorganisms was established at our institute, which is recognized by the World Federation of Culture Collections (WFCC). This effort assumes considerable importance from the point of conserving microbial biodiversity. conserving microbial biodiversity.