28 Groundwater Microbiology

Dr Tejpal Dhewa

Learning objectives:

• To understand the status of Groundwater in world
• To understand the various environments (i.e. Physical, Chemical and Biological) of Groundwater
• To gain knowledge about Groundwater contamination: major sources and entry points The understand the Microbial communities and their interactions.

Groundwater Microbiology

TABLE OF CONTENTS

1. Introduction

2. Physical and Chemical environment 2.1 Physical environment

2.2 Chemical environment

3. Microorganisms in Groundwater

3.1 Groundwater contamination: major sources and entry points

3.2 Microbial diversity

3.2.1 Bacteria

3.2.1.1 Gram-Negative bacteria

3.2.1.2 Gram-Positive bacteria

3.2.2 Viruses

3.2.3 Archaea

3.2.4 Protozoa

3.2.5 Fungi and helminths

4. Microbial processes

4.1 Ammonia oxidation

4.2 Denitrification

4.3 Manganese, iron and sulphate reduction

4.4 Dehalogenation

5. Summary

1.Introduction

Groundwater is one of human’s most important source for life, provide about 0.63% of the world’s total water and 20% of the available fresh water resources (Fig.1). The surface water is recharging groundwater systems by its downwards movement due to gravitational forces through soil and sediment layers, unsaturated zones that typically consists of tiny pores and cracks in the soil, gravel and then finally reaches to a stable water saturated layer called the water table. The groundwater or aquifer is underneath the water table (Fig. 2). These protective layers act as effective mechanical and biological filters and thus, providing a natural clean-up of newly produced aquifer. That is why in most of countries globally it is used as the principal source of drinking water for example, Norway, Bremen, Hamburg, Saarland and Schleswig-Holstein are 100% groundwater consumer states, India more than 95% of drinking water comes from aquifers and whereas Germany 70%, Berlin 25% and Europe only 13% groundwater usage. Naturally, diverse natural microbial populations playing a significant role in self-purification of groundwater but subsequent microbial (bacteria, protozoa and viruses) and chemical contamination due to anthropogenic activities may affect the drinking water quality.

2.Physical and Chemical environment

The physical and chemical environment of aquifers is quite simple and discussed below:

2.1 Physical environment

Aquifers are geological formation, which can store and transmit water. Normally, following types of aquifers exists (Fig.3):

• Confined aquifer: confined in between relatively impermeable material like clay, normally recharged indirectly through infiltration or directly by underground springs.
• Unconfined aquifer: shallow, open to the atmosphere, bounded on bottom by impermeable material and recharged directly and indirectly by rainfall.
• Semi-confined aquifer: leaky aquifers are confined by semi-permeable materials.

Normally, the temperature of these aquifers is relatively stable because the specific heat capacity of water is high and also due to the rock, soil and other layers protect aquifer from heat fluctuating with the climate.

2.2 Chemical environment

Typically, the mineral content in the aquifer is constant. However, these could be higher in the surface water from the same area. Commonly, Mn2+, Fe2+, H2S, CO2 and chlorinated solvents are found, concentrations of silica and nitrates could be quite high but no dissolved oxygen (DO).

3.Microorganisms in Groundwater

Groundwater is an active and quantitatively very important constituent of the hydrological water cycle. Microorganisms including pathogens present in surface water and soil are regularly or at least less frequently enter aquifers through below discussed possible routes.

3.1 Groundwater contamination: major sources and entry points

• The possibility of groundwater contamination is determined by the survival and transport rate of pathogens within the system. Following factors affect the growth, survival and transport rate of pathogens (Fig.4 & 5):
• Environmental conditions
• Hydrology of aquifers
• Physico-chemical properties of water and soil in the system
• Microbial characteristics and physiological condition of the organisms

The environmental condition like temperature affects the death rate of organisms, particularly viruses. Higher temperature has increased death rate of the microorganisms whereas lower temperature has lower death rates. In addition, temperature also affects the viscosity and density of water, resulted change in water velocity. Rise in temperature will also cause better molecular diffusion. The composition of water and hydrology affect the water velocities and direction of flow in ground.

Organisms usually survive longer in such systems if organic matter is readily accessible, as it can be a source of nutrients for the microorganisms. Though, antibiotics and other toxic organic materials will reduce the growth and survival rate of pathogens. Microorganisms flourish in moist soil due to water as a substrate and facilitate nutrient transport, over and above transport of organisms within soil layers. Remarkably, the pH of surroundings is playing very important role in dissolution of compounds and charge distribution in system that affects the adsorption of microorganisms onto the soil (Fig.5). However, adsorptions are also influenced by soil type, texture, pore size etc.

The most important characteristics of the organisms are their potential to compete for available substrate and nutrients. For example, microorganisms will have a shorter survival time if predators exist there or any competing organism present. In such conditions carbon sources become insufficient.

The transport of microorganisms is also limited by the population size and the soil pore space, though the microbial population growing in floc will have higher probabilities of survival due to the protection and chances to share resources within the floc but reduced the transport rate because of blockage in pores.

Scientists have endeavoured to predict the transport of organisms in groundwater using various mathematical models, which are based on following two transport mechanisms (Fig.6):

1. Advection: the transport of microorganisms by water in the direction of flow and it is regulated by the average linear velocity of the groundwater. In this mechanism, organism movement is directly proportional to the velocity of groundwater. Rise in groundwater velocity will result in more organism’s movement.
2. Dispersion: the transport of organism carried out by water in direction other than that of the flow. It is combined effect of mechanical mixing and sum of molecular diffusion. A number of factors including pore size, path length and friction in pore affect the scattering of organisms or other contaminants in the ground (Fig.7).

Growing population, urbanization, industrialization and expending land use are significantly contributing wastes in the environment. In developing countries, most of the domestic and industrial  wastes  are  not  treated  properly prior  to  discharge  in  natural  water  resources. Usually, wastes including fecal materials (originating from human and animal feces) are potential  source  of  the  pathogenic  microorganisms  such  as  bacteria,  viruses,  protozoa, helminths etc. These microorganisms can contaminate groundwater by following way (Fig.8):

• Improper sewage or effluent disposal systems
• Leaking sewers and overflowing septic tank
• Underground storage tank
• Accidental (waterborne outbreaks) and non-accidental wastewater discharge Animal manure and compost
• Use of untreated or partially treated sewage sludge into agricultural fields as fertilizer.
• Surface water contamination through non-point sources or with treated or untreated effluents, agricultural runoff and sometimes cross contamination

3.2 Microbial communities

Microbial communities are surprisingly diverse in this environment (Fig.9). According to studies, bacteria are estimated to be the principal microbes exist in groundwater systems, followed by protozoa, archaea and some fungi. Remarkably, the microbial communities are expected to include heterotrophs and chemolitho autotrophs, which are attuned to living in nutrient-deficient and anaerobic conditions, particularly for deep and confined aquifers. Thus, the microbial communities appear in aquifers are oligotrophs and k-strategists that reproduce slowly and have low tolerance to rapid changes. Lithoautotrophs are more likely to be more important in deeper aquifer habitats, where oxygen is almost not available and inorganic electron donors are more like present than readily degrading organic materials. Moreover, autotrophic methanogens and acetogens are also commonly found in relatively larger numbers in deeper aquifer environments. Although, it is anticipated that the diversity of most of such organisms is mostly restricted to shallow, unconfined aquifers. Thus, aquifers are considered extreme habitats due to absence of sun light, low availability of organic carbon and other nutrients, low constant temperatures make them extremophiles that are well-adapted to such conditions. 

3.2.1 Bacteria

Groundwater near the land surface normally crowds with microbial population. Bacteria, which are microscopic, unicellular prokaryotes, are most abundant than any other organism in the soil and groundwater. About 100 million to 1 billion bacteria per gram of dry soil recovered from nearby of plant roots. These number decreases extremely with soil depth below the root zone due to nutrient shortage. However, bacterial population have been detected in core samples from a depth of 2.8 kilometres below the earth’s surface and even at a depth of 3.2 kilometres in gold mines of South Africa. The total number of bacterial population exists in groundwater ecosystems may depends on several factors. Naturally, the average bacterial population found between 102 to 106 cells per cm3 in groundwater and between 104 to 108 cells per cm3 in sediment. However, these numbers may increase if aquifers get contaminated. Bacterial pathogens transmitted by the fecal oral routes are detected in groundwater (Table 1).

3.2.1.1 Gram-Negative bacteria Gram-negative    bacteria    like     Azotobacter,    Acinetobacter,    Neisseria,    Moraxella     and Pseudomonas are found extensively in groundwater systems. According to studies at a site in South Carolina, aerobic and chemoheterotrophic bacteria exist in deep aquifers and other subsurface sediments. Out of 95% recoverable colonies of nonstreptomycete bacteria, more than 80% were gram-negative rods.

3.2.1.2 Gram-Positive bacteria Gram-positive bacteria are not abundant in groundwater systems. However, many important human pathogens like Streptococcus, Micrococcus and Staphylococcus exist frequently.

3.2.2 Viruses

The transport of viruses through soil to aquifer and then to drinking water has been a topic of a great fear. In past, several epidemics of infectious diseases have been reported due to drinking of the contaminated groundwater. Since, viruses are smaller acellular particles that may easily pass through filters like soil and sediments and therefore, the penetration of pathogenic viruses to groundwater systems seems much more expected than for bacteria and protozoa. Numerous factors including soil type, pH, conductivity of percolating water, dissolved organic contents and flow of water are playing significant role during the viral transport through soils. According to studies, viruses originating from wastewater infiltration units are able to travel horizontally in aquifer for hundreds of meters and vertically up to 67 meters. The risk of viral contamination of water is further increased due to the shedding of enteric viruses into the environment. It is estimated that about 65% of the waterborne diseases are caused by enteric viruses. In addition, viral pathogens from feces have much longer survival times in water than most intestinal bacteria and protozoa and are also resistant to common disinfectants used during disinfection of water prior to drinking. Such features make pathogenic viruses the most vital entity for fecal contamination of aquifers. As of now, more than 15 different groups of enteric viruses including more than 140 different serotypes exist in human gut and serves as waterborne pathogen.

3.2.3 Archaea

Archaea have been found throughout depths of several thousand meters in groundwater. Typically, methanogenic and oxidizing Archaea are two main groups found in groundwater. Methanogens are a distinctive functional group within the Archaea. They found in anaerobic aquifers where they conduct terminal respiratory reactions along anaerobic degradation pathways in the absence of alternative electron acceptors. In a study of Swedish granite aquifer, Methanosarcina-like organisms were reported in groundwater from depths of 45-68 meters. In addition, Methanohalophiles sp. and new alkaliphilic Methanobacterium sp. were also recovered from various depths. Methanosaeta sp. has often been reported from various contaminated sites.

3.2.4 Protozoa

Protozoa such as amoebae, flagellates and ciliates unicellular eukaryotic organisms are also common in groundwater. Typically, protozoa are much larger than bacteria and several types in groundwater feed on bacteria. In general, protozoa are useful in groundwater to maintain the bacterial density but can be harmful to human beings if ingested. For example, protozoan parasites including Giardia lamblia is a waterborne pathogen found in contaminated groundwater and its cysts enters the water supply through fecal contamination and causes giardiasis.

3.2.5 Fungi and helminths

There is little information available on the existence of pathogenic fungi in groundwater. Though, it is quite possible that most fungi are removed by chemical coagulation with iron or sand filtration during water treatment process and fungal spores are destroyed with chlorine dioxide and ozone through disinfection process. Although, waterborne parasitic helminths such as Ascaris lumbricoidides are commonly found in surface water of developing countries but their transmission into groundwater is doubtful due to the size of organism and their eggs are easily removed from contaminated water by filtration during purification of drinking water.

4.Microbial processes

As aquifers are featured by no light, low organic carbon and nutrient availability, therefore most of the microbial communities found in groundwater need to use inorganic electron donors and light-independent chemical reactions as a source of energy. In sediments, most of the microbes would be lithotrophs whereas in shallow, unconfined aquifers, aerobic organotrophs could be present. Following microbial processes are useful in cleaning of groundwater:

4.1 Ammonia oxidation

Ammonia oxidation is carry out by both bacteria and archaea in the groundwater. However, the rate of ammonia oxidation is limited by the available oxygen. In this process, ammonia is oxidized into nitrite and nitrate and the finally be removed from the system by reduction to dinitrogen by denitrification.

4.2 Denitrification

In groundwater, certain bacteria such as Bacillus, Pseudomonas and Burkholderia play significant role in denitrification. This process remove nitrate (main groundwater pollutant leached from fertilizer amended agricultural regions) from groundwater. Some inorganic compounds like FeS2 and Fe [II] – silicate may also be utilized by these microorganisms.

4.3 Manganese, iron and sulphate reduction

As there is no dissolved oxygen is available in groundwater. Therefore, anaerobic conditions are dominant in groundwater. In anaerobic zone, microbial populations could perform decomposition coupled with iron manganese, and sulphate reduction. For example, Thiobacillus ferroxidans, Shewanella putrefaciens, Leptothrix, Desulfovibrio desulfuricans etc. Desulfovibrio desulfuricans is well adapted to the specific salt content and temperature of the groundwater. Ground H2S, S, S2- and other sulphur compounds including tetrathionate, thiosulfate and sulphites can be oxidized by the genera Beggiatoa, Crenothrix, Thiobacillus and Thoploca. Certain chemoorganotrophs can perform anoxic reduction of Mn4+ to Mn2+. Such microbial processes determined by the redox conditions and could affect carbon, sulphur and iron cycle in the groundwater.

4.4 Dehalogenation

Synthetic halogenated organic compounds in the groundwater could persist for very long duration and considered as a serious health issue. Halogens can be removed from organic compounds by dehalogenation. Interestingly, anaerobic dehalogenation reaction can efficiently degrade a wide variety of halogenated contaminates in groundwater.

5.Summary

Groundwater is the most important resource of freshwater available on the earth, more than 95% of the worlds available freshwater (except glaciers and ice caps) is underground. Normally, it is well secured by covering of soil and sediment layers and used for drinking purpose as it is free from contamination. Growing populations, industrialization and urbanization are the key sources of groundwater contamination. Though, prior to the 1970s, a common supposition in society was that groundwater was the most pristine water and did not have any microbial population. This was somewhat supported by the limitations of sampling methods and culture based techniques. However, new culture-independent (molecular) techniques including direct extraction, cloning and community fingerprinting etc. have allowed scientists to begin to explore microbial communities exists in groundwater systems. Metagenomic approaches are likely to foster groundwater microbial diversity. In general, groundwater ecosystem deals huge and complex habitats for diverse microbial communities, which critically linked to groundwater self-purification and storage at high quality for decades and centuries. The microbial content is influenced by changes in the environment including pollution of groundwater, percolation, pH, salt content, organic content etc. The key microbial groups identified in aquifer systems are members of proteobacteria, bacteriodes, actinobacteria, firmicutes, Euryarchaeota and crenarchaetota were also identified. In addition, few invertebrate communities have also been reported in groundwater habitat between 100 to 1000 species. For example, members from phylum annelida, chordate, cnideria, gastropoda, nematode, profera and arthropoda. Several abiotic and biotic factors including local hydro geochemistry, dispersal of microbes from overlying unsaturated zones, food-web structure in groundwater systems and availability of oxidable substrates may directly or indirectly control microbial diversity in groundwater systems. In spite of the recent developments, groundwater microbiology is still a topic that has not been fully discovered and hence, inadequate information is available.