Bacteriological Profile and MAR Index

Biofilm and MAR index of bacterial isolates from drinking water distribution system 

Biofilm in the drinking water pipelines have been considered as the primary factors in degradation of the physico-chemical and microbiological parameters of drinking water. The study has shown that the isolates from the biofilm of drinking water distribution system has Multiple Antibiotics Resistance index of more than 2. 

Microbiology of Drinking Water Distribution Systems

Bacteria in biofilm of drinking water distribution system 

Biofilm in the drinking water pipelines have been considered as the primary factors in degradation of the physico-chemical and microbiological parameters of drinking water. The study has shown that the isolates from the biofilm of drinking water distribution system has Multiple Antibiotics Resistance index of more than 2 of the isolated bacteria. 

Factors affecting Biofilm Growth

According to Momba et al., (2002) several factors promote bacterial growth in drinking water distributions systems, called regrowth, which occurs in the water phase and in biofilms on the pipe surfaces and reservoirs. Different factors that affect regrowth in drinking water distribution systems are as follows:

 

1. Temperature and pH

Temperature and pH are major factors that affect the microbial growth by modifying the electrostatic interactions between surfaces and microorganisms, enzymatic reactions, and many other properties e.g. diffusivity, solubility. Microorganisms do withstand some variations in the pH as their own metabolic activities alter the pH in the vicinity by producing acids. But bacteria do have their optimum pH. Chen et al., (2005) in his recent study concluded that attached Pseudomonas fluorescens accumulated at a greater extent and more cohesively in the biofilm at neutral pH than in other acid or basic media.

2. Disinfectant Agents

During transportation bacterial regrowth is efficiently prevented by using chemical disinfectant and by maintaining the residual in distribution system. However, regrowth do occur when residual decay further down in the distribution system. Biofilm matrix along with EPS enclosed the bacteria and protects from the disinfectants by preventing the penetration of biocides, limiting the diffusion or by reacting. Lower concentration of disinfectant concentration was found within the biofilm than in the water. Besides, it actuated first in the outermost layer where as bacteria were found to be metabolically active in the inner layers. Induction of stress responses and development of biofilm-specific biocide resistant phenotype may contribute to biocide resistance (Huang et al., 1995). Pathogenic bacteria hosted inside the protozoa were also found to be one of the ways to increase the resistance against biocides (LeChevallier et al., 1988)

3. Availability of nutrients

Drinking water distribution systems is an oligotrophic environment with low contents of carbon, nitrogen and phosphorous. Several reports from the drinking water distribution systems in Australia (Chandy and Angles, 2001), France (Servais et al., 2004), Singapore (Hu et al., 2005), Netherlands (van der Kooij, 1992) and China (Bai et al., 2006) observed that the organic carbon content was the limiting nutrient because an increase in this nutrient promoted bacteria regrowth while in other studies conducted in Japan (Sathasivan and Ohgaki, 1999) and Finland (Lehtola et al., 2002) phosphorus had been found as limiting nutrient. Thus, nutrient availability, impact on structure, sloughing rate of biomass, EPS production and microbial adhesion of biofilm (Veiga et al., 1997).

4. Hydrodynamic Conditions

In drinking water distribution system, the hydrodynamic conditions may ranges from laminar to turbulent flow, however stagnant (no-flow) water also occurs in places where water consumption is low and in reservoirs of buildings. The hydrodynamic condition may cause different effects on biofilm accumulation and detachment. Increase in flow velocity initially increases the nutrient transport rates until it reaches maximum and then decreases with the further increase in flow velocity. Besides, the flow velocity increases the biofilm density and detachment. It has been found from previous research that hydrodynamic conditions and the nutrients are the two major factors that influence biofilm growth, its structure, density and thickness. Higher flow velocity have been found to increase cells hydrophobicity that will favor cells aggregation and hence biofilm accumulation (Liu and Tay, 2001; Liu et al., 2003).

5. Surface Material

In drinking water distribution system, Iron-based, cement-based materials and polymeric materials such as PVC (Polyvinyl chloride) and PE (Polyethylene) are used in distribution network pipeline. In iron-based pipeline materials, corrosion have been found as the major factor that increases the soluble iron in the water, transport head loss and turbidity (McNeill and Edwards, 2001).

There is still some controversy about the effect of surface materials on biofilm development. Some researchers (Momba and Kaleni, 2002) demonstrated that drinking water biofilms grew less on polymeric materials (PE, PVC,  Teflon) than on iron materials (grey iron, cast iron, galvanized steel, stainless steel, cemented steel, asbestos-cement and cemented cast iron) however, opposite results were found by other researchers (Cloete et al., 2003; Bachmann and Edyvean, 2005) where as in other works (Zacheus et al., 2000; Wingender and Flemming, 2004) no significant difference was found.

Roughness, corrosion resistance, hydrophobicity and hydrophilicity, Migrating components from surfaces and valves and joints materials of the pipe materials used in drinking water distribution system has been identified as an important factor affecting biofilm formation (Pedersen, 1990). In a recent study it had been found pipe service age was an important factor in the consumption of chlorine and this effect decreases in the following order cast iron > steel > cement-lined cast iron = cement-lined ductile iron > PVC = PE (Al-Jasser, 2007).

 6. Protozoa Grazing

Protozoa are considered the major organisms responsible for bacterial grazing in aquatic environments, which has been shown to limit biofilm accumulation in drinking water systems (Berry et al., 2006; Snelling et al., 2006). In contrast to predation, association of several pathogenic bacteria to protozoa has been found as the latter have high resistance to chlorine; hence promoting resistance against disinfectants and increasing the health risk events.

Biofilm Formation in drinking water distribution

Biofilm Formation in drinking water distribution

1. Formation of a surface conditioning film

It is a thin layer of organic molecules and ions covering the adhesion surface and is formed before attachment of microorganisms. Physical or chemical adsorption helps these processes. The former is a reversible process and includes nonspecific bonds such as van der Waals with low adsorption energies. Whereas latter is a nonreversible and includes specific chemical bonds such as electrostatic, covalent and hydrogen bonds, dipole interactions, and hydrophobic interactions with higher adsorption energies. In this process several adsorbed molecular layers are formed which determines the strength of the biofilm adhesion. Besides, the conditioning film helps bacteria to attach on the surface of pipeline by neutralizing flow velocity and do also provide nutrient for bacteria (Marshall, 1996).

2. Initial adhesion of “pioneer” microorganisms to the surface;

Planktonic (free floating) microorganisms are transported towards the surface either by fluid dynamics, gravitational forces and Brownian motion, or by migration through active cell motility (e.g. flagella). The surface electrostatic charge and hydrophobic interactions are also found to affect this approaching and adhesion (Mueller, 1996).

The first arriving free floating bacteria adhere to the surface initially through weak, reversible electrostatic attraction and van der Waals forces. If not immediately separated from the surface, the bacteria are anchored more permanently by developing stronger bonds for the attachment to the surface for example, by active cell biosynthesis of EPS and by chemical forces. These cells become irreversibly adsorbed. Different mechanisms had been proposed to explain the bacteria attachment to surfaces with different hydrophobicity character (Marshall, 1996).

3. Biofilm growth

The attached bacteria start to grow, excrete organic polymers, and initiate the formation of the biofilm matrix. Tolker-Nielsen et al., (2000) observed that the first micro-colonies formed were mono-species. Further attachment of planktonic bacteria and of inorganic particles will contribute to a structurally heterogeneous biofilm growth, as well as, migration of attached bacteria between and inside of micro-colonies. During this phase, bacteria detachment events occur although at a lower extent compared to the growth rate.

4. Biofilm maturation - equilibrium between accumulation and detachment.

Mature biofilm are composed of an organized consortia of microorganisms embedded in an organic matrix that protects the bacteria. The structure of a mature biofilm depends on the microbial composition, EPS production, the nutrient availability, hydrodynamic conditions and temperature. In a biofilm several processes may occur simultaneously: bacterial detachment into water, attachment of planktonic bacteria, growth, death etc. In a mature biofilm these processes are at equilibrium and the attached cells per unit surface area are constant with time although with periodic fluctuations. At this phase, the biofilm reach the highest thickness that does depends on the hydraulic conditions, the mass transport and the biofilm cohesion (Momba et al., 2002).

According to Momba et al., (2002) several factors were observed to promote detachment of biofilm portions or of isolated bacteria. These factors are the following:

Sloughing off: The increase of shear stress, alternating flow conditions and abrasion due to particle collisions promote sloughing off of biofilm pieces that were not well cohesive.

Starvations of bacteria: It does promote size reduction, and increase of bacteria fragmentation and of motility which will increase biofilm detachment.

The increase of nutrients: it promoted the release of bacterial cells up to 80 % the total attached ones. This biofilm dispersion phenomenon was associated with increased expression of flagella genes.

Chelating agents: A chemical change in the EPS due to presence of chelating agents (Ca²⁺) that will reduce the cohesive strength of the attached cells.

Surfactants: The excretion of surface-modified products (surfactants) by certain bacteria may promote detachment.

Signaling molecules: In biofilms the excretion of certain signaling molecules induced the detachment events.

Biofilm formation inside water distribution systems

Biofilm formation inside water distribution systems

A biofilm is a surface deposit of bacteria, other microorganisms, and organic and inorganic materials that accumulate within a slime layer. Biofilms can form on solid and liquid surfaces when nutrients and water are present. Biofilms can form inside drinking water distribution systems and can sometimes cause a number of problems.

Biofilm Formation
Distribution systems are complex environments that can provide many opportunities for biofilm development. This development may occur fairly rapidly or slowly, sometimes over a period of years. However, clean pipes (especially metal pipes) are not initially attractive surfaces for bacteria.

Bacteria are typically the first microorganisms to colonize pipe surfaces. Once enough organic material adheres to the pipe surface — a process referred to as “conditioning” — bacteria can begin to attach.

Once the bacteria reach a critical density, they begin to produce a gelatinous substance that gives biofilms their characteristic slimy nature. This slime layer makes up the majority of the weight and volume of the biofilm. After the slime layer forms, a veritable micro-ecology can flourish. The slime layer helps trap additional organic particles that many bacteria can use for food and energy.

Other microorganisms including viruses, protozoa, algae, fungi, and helminthes may become associated with or entrained within the biofilm. Some protozoa graze on biofilm bacteria creating a food web.

Biofilms provide a number of advantages for attached organisms compared to free-floating (planktonic) organisms. In a low-nutrient environment, it is easier for microbes to let the nutrients come to them rather than to search for the nutrients. The slime layer allows metabolic byproducts or wastes to accumulate, some of which may be used as food by other microorganisms, forming a cooperative ecology.

The biofilm also protects the inhabitants from the effects of disinfectants — biofilm microbes are many times more resistant to disinfection than planktonic microbes. Biofilm thickness is variable but is usually in the range of 50 to 100 microns. As the thickness increases, pieces of biofilms can shear off, allowing for colonization of downstream sections of the system.

Microorganisms can enter the distribution through two main categories:

• Surviving the treatment process
• Recontamination

Most microorganisms found in distribution systems biofilms are also found in the system’s source water. They may survivedue to ineffective treatment such as filter breakthrough or ineffective primary disinfection. However, even effectively treated water contains some bacteria in small numbers. Potable water is not sterile.

Assuming water of good quality enters the distribution system, there are still numerous ways the water can be contaminated, including cross connections and back flow. Leaking pipes, joints, and valves can also allow for the entry of microbes, especially during temporary periods of negative pressure.

Poorly designed or maintained finished water reservoirs and tanks can allow for recontamination if birds and other animals, including humans, have access.

Repairing and replacing distribution system components also allows for the introduction of microorganisms if care is not taken to disinfect repaired or replaced mains and tools introduced into the system, such as mobile cameras.

Some biofilm organisms can also accelerate the corrosion of some types of pipes. Iron-oxidizing bacteria oxidize iron and steel, depositing iron oxides (rust) in raised deposits called tubercles. Sulfur-oxidizing and sulfurreducing bacteria produce sulfuric acid and hydrogen sulfide, respectively, which can cause pitting of pipe surfaces. Corrosion products, such as iron oxide sediments and tubercles, provide additional habitats and attachment sites for other biofilm organisms.