Rakeshkumar M. Jain 1 Ph.D. Thesis

1. INTRODUCTION

The diversity of microbial habitants and their ecological and metabolical roles are

being explored across a wide range of natural environments: soils, air and water.

Microorganisms reside in almost every possible environments including some of the

most extreme habitats such as the deep-sea vents, hydrothermal vents, volcanic areas,

thermal springs, Arctic and Antarctic sea ice, salt lakes, marine salterns, metal-

contaminated sites, soda lakes, sea sediment, alkaline soil, sodic soil, soda ash, food,

beverages, chlor- alkali, textile industry, hydrocarbon or oil contaminated alkaline

waste-waters and sludge (Horikoshi and Grant, 1998; Desai et al., 2004; Celso et al.,

2007; Pollock et al., 2007; Joshi et al., 2008; Poli et al., 2011). Extreme environmental

conditions include high temperature, pH, pressure, salt concentration, low temperature,

pH, nutrient concentration and water availability, as well as conditions having high

levels of radiation, harmful heavy metals, toxic compounds, oil and organic solvents

contaminated sites (Horikoshi, 1996; Satyanarayana et al., 2005). Extremophiles are

valuable resources in biotechnology as they are source of enzymes (extremozymes),

drugs and biomolecules (proteins, biopolymers and organic acids) with enormous

stability in extreme conditions (Horikoshi, 1999). Extremophiles opened a new

frontier of science especially in biotechnology and bioremediation which are major

growing and reliable fields, providing the green technologies for the future.

Alkaliphilic bacteria are a subset of extremophilic microorganisms that grow

optimally at pH values above 9, often between 10 and 12, but cannot grow or grow

slowly near neutral pH (Grant et al., 1990; Horikoshi, 1999). Alkalitolerant is an

organism capable to grow or survive at pH values more than 9.0 but its optimum rate

of growth is around neutrality or less. Alkaliphiles consist of two main physiological

1. Introduction

Rakeshkumar M. Jain 2 Ph.D. Thesis

groups of microorganisms; alkaliphiles and haloalkaliphiles. Alkaliphiles require

alkaline pH whereas haloalkaliphiles require both, alkaline pH and high salinity for

their optimal growth (Grant et al., 1990). Alkaliphiles having salient feature associated

with the cell surface, which discriminates and maintains the intracellular neutral

environment apart from the extracellular alkaline environment. The peptidoglycan

layer in alkaliphiles has excess of hexosamines and amino acids having higher cross-

linking rate at higher pH values; providing shielding effect by tightening the cell wall.

Moreover the cell wall contains certain acidic polymers, viz. galacturonic acid,

gluconic acid, glutamic acid, aspartic acid and phosphoric acid which may provide cell

surface its ability to absorb sodium and hydronium ions and repulse hydroxide ions

that supports the cells to grow in alkaline environments (Horikoshi and Grant, 1998;

Horikoshi, 1999). Alkaliphilic microorganisms grow luxuriously at pH 9.0 to 11.0 and

require Na+ ions for growth. The intracellular pH regulation is dependent on the

presence of sodium, which switches over from cytoplasm into the medium by H+/ Na

+

antiporters (Krulwich et al., 1997). In Na+ dependent transport systems, the H

+ is

exchanged with Na+ by Na

+/H

+ antiporter systems, thus generating a sodium motive

force, which drives substrates and Na+ both into the cells. Not only controlling the

protons, but re-entry of Na+ into the cell is maintained in Na

+-dependent pH

homeostasis. Na+-coupled solute symporter and Na

+-driven flagella rotation ensure a

net sodium balance. The combined action of antiporters coupled with respiration

provides the cell a means of controlling its internal pH (Krulwich et al., 1994;

Horikoshi and Grant, 1998; Satyanarayana et al., 2005). Alkaliphilic microbes offer a

multitude of potential applications in various fields of biotechnology. Since the

discovery of serine protease (alkaliphilic enzyme used in detergent additives) in 1970,

a large number of alkaliphilc exoenzymes are made available such as, alkaline-

1. Introduction

Rakeshkumar M. Jain 3 Ph.D. Thesis

protease (detergent industry, decomposing the gelationous coating of X-ray films for

silver recovery, dehairing), amylase (for hydrolyzing starch into glucose), pectinase

(degumming of ramie fibers, improving paper quality), pullulanase (dishwashing

detergents), cellulase (laundary detergent additives), alginase, catalase, RNase, DNase,

restriction enzyme, β1,3-glucanase, xylanase, β-galactosidase, α- galactosidase,

penicillinase, maltose dehydrogenase, urease, polyamine oxidase, β-mannanase, β-

mannosidase, lipase, chitinase and cyclomaltodextrins glucanotransferases (Horikoshi

and Grant, 1998; Horikoshi, 1999; Menon et al., 2010).

Bioactive compounds (phenazine and others) produced by alkaliphiles are

effectively used as pharmaceutical compounds. Many strains exhibit antimicrobial

activity against Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus,

Mycobacterium smegmatis and Candida albicans. Apparently, alkaliphiles produce

important metabolites like 2- phenylamine, carotenoids, cholic acid derivatives,

organic acids, antibiotics, enzyme inhibitors and possess siderophoric activity

(Horikoshi, 1996; Horikoshi and Grant, 1998). From early 1971, studies of

alkaliphiles have revealed many novel biomolecules with tremendous potential in

biotechnology (Horikoshi, 1996; Nicolous et al., 2010).

Rapid industrialization, technology development and increase in population

boosted environmental pollution, especially the aquatic environment with a multitude

of contaminants. India is facing serious environmental management crisis, particularly

with respect to pollution of soil, air and water (Chakraborti et al., 2011; Thavasi et al.,

2011). Our requirements for resources are ever increasing. This has led Einstein to

quote:

“Resources when not used in the right place is called pollution”

1. Introduction

Rakeshkumar M. Jain 4 Ph.D. Thesis

Gujarat is the second most industrialized state in India and is the largest

producer of salt in the country. It contributes 70% of its total salt production. It has a

coastline of about 1660 km and most of the industries are located along river basins,

estuaries and coastal areas. Major problem faced in textile, paper and pulp, soda ash,

beverage and chlor-alkali industries is the treatment of huge volume of wastewaters

having extremely high pH and salinity. These wastewaters have high pH, ranging from

8.0-12.0, hence not permitted to drain out as such. Common bioremediation processes

use terrestrial microbes which are unsuitable for the wastewaters having high salinity

and alkalinity hence, prior neutralization is essential as most of the microbial strains

present in activated sludge process work well near neutral pH. Alternatively, isolation

and utilization of microbes, which are best suited to the extreme conditions of salinity

and pH would help in providing an economic and safe process for neutralization of

alkaline waste-waters. Generally, neutralization of alkaline waste-waters is being done

purely by chemical means where a huge amount of acid is used which is neither

economically feasible nor safe as it poses serious health hazards. The biological

treatment can be used as an alternative to chemical processes as this can be cost

effective, environmental friendly and publicly accepted technology (Kumar et al.,

2005a, b; Kumar and Kumar, 2008; Dafale et al., 2010; Kulshreshtha et al., 2010;

Yang et al., 2011).

The use of alkaliphiles producing biosurfactants would be suitable option for

bioremediation of petroleum hydrocarbons contaminated alkaline environment.

Biosurfactants are heterogenous, complex and structurally diverse group of surface

active agents, produced by living microorganisms from different habitats, which either

adhere to cell surface or excreted from the cell (Mulligan, 2005; Parikh and

Madamwar, 2006; Kumar et al., 2007; Thavasi et al., 2011). Biosurfactants are

1. Introduction

Rakeshkumar M. Jain 5 Ph.D. Thesis

amphiphilic compounds (with polar and non polar moieties) comprised of glycolipids,

lipopeptides, lipoproteins, fatty acids, neutral lipids, phospholipids, particulate and

polymeric compounds (Desai and Banat, 1997; Banat et al., 2000; Bramhachari et al.,

2007). These amphiphilic compounds contain a hydrophobic and a hydrophilic moiety,

and have the ability to reduce interfacial and surface tension between different fluid

phases. The hydrophilic moiety of a surfactant is defined as the “head”, while the

hydrophobic one is referred to as the “tail” of the molecule which generally consists of

a hydrocarbon chain of varying length (Figure 1.1) (Banat et al., 2000; Bramhachari et

al., 2007).

Figure 1.1: Biosurfactant is amphiphilic in nature.

Biosurfactant production is being done using conventional (sugars, acids,

alcohols) and unconventional (agroindustrial waste) raw materials. Limited production

at large scale has been realized for many biosurfactants due to expensive raw material,

low production yield and high purification cost. The main factors limiting microbial

biopolymer are associated with their production costs, mainly substrate cost. Carbon

substrate is an important limiting factor, affecting the production by influencing its

1. Introduction

Rakeshkumar M. Jain 6 Ph.D. Thesis

quality and quantity (Das et al., 2009). Utilization of unconventional raw materials

make the process economical and ecofriendly (Makkar et al., 2011).

One of the strategies to improve production is to optimize the growth media in

order to get maximum production (Mukherjee et al., 2006). Formulation of an

optimized production medium involves selection of the appropriate nutrients at their

accurate levels to provide an ideal microenvironment to support growth and metabolite

production. In statistical experimental design, response-surface methodology (RSM),

has been used to optimize media components to increase yield of the product (Sen,

1997; Sen and Swaminathan, 1997; 2004).

Biosurfactants have different chemical structures, compositions and a wide

range of biotechnological applications in dairy, food, beverage, cosmetics, detergent,

textile, paint, mining, petroleum, paper pulp and pharmaceutical industries (Cameotra

and Makkar, 2004; Singh and Cameotra, 2004; Bhaskar and Bhosle, 2005; Rodrigues

et al., 2006). In recent years, interest in the exploitation of valuable biosurfactants has

been increasing for various industrial applications and the attention towards

biosurfactant producing extremophilic bacteria has greatly enhanced (Rodrigues et al.,

2006; Nicolaus et al., 2010). Extremophiles are of interest because of their potential

for environmental applications, especially as source of emulsifiers for bioremediation

of hydrocarbon and toxic metal from contaminated soils and wastewater in extreme

conditions (high pH and salinity) (Urum and Pekdemir, 2004; Mulligan, 2005; Whang

et al., 2008; Calvo et al., 2009; Lai et al., 2009; Nicolaus et al., 2010; Satpute et al.,

2010; Thavasi et al., 2011). They are also effective in a wide range of extreme

conditions including temperature, pH and salinity as compared to chemical surfactants

and commercial detergents for removal of hydrocarbons from soil, waste water and

cotton cloth (Banat et al., 2000; Mulligan, 2005; Bramhachari et al., 2007; Mukherjee,

1. Introduction

Rakeshkumar M. Jain 7 Ph.D. Thesis

2007). Use of biosurfactants is ecologically accepted due to their specificity, high

solubility, less toxicity and biodegradable nature (Satpute et al., 2010; Thavasi et al.,

2011).

The present study was undertaken towards application of alkaliphilic bacteria in

bioremediation with the following objectives:

1. Isolation of alkaliphilic/ haloalkaliphilic bacteria.

2. Studies on pH and salt tolerance of isolated bacterial cultures.

3. Identification of isolated bacterial cultures.

4. Neutralization of alkaline industrial wastewaters using potential alkaliphilic

bacteria.

5. Assess the potential of isolated alkaliphiles for biosurfactant production and

optimization of conditions for maximum production.

6. Application of biosurfactants in bioremediation of oil.