Chronological Sequences in the Biofouling Process

There are a number of stages which commonly occur during the process of biofouling in a water well. These stages are affected by a number of major factors which include the water quality and production rate, the nature and porosity of the media around the well, the design and construction of the well and the degree of preventative maintenance which is being applied during the operation of the well. There remains, however, a sequence of events which may commonly occur and these are described below:

1. Initial Colonization. It is to be expected that the natural (intrinsic) and introduced (extrinsic) microorganisms will be present within the environment affected by the installation of the well. The intrinsic flora will have arisen from the groundwater, the unsaturated media above the water table, and from the soil. The extrinsic microorganisms are those introduced to the environment by the introduction of drilling equipment, waters, personnel, muds and various chemicals required for the construction and development of the well. These microorganisms will compete for the available surfaces onto which they attach. Desirable surfaces include those which are charged, occur at the redox front, where turbulence is being generated in the water, are approximate to nutrient supplies (e.g., organic deposits, higher concentrations of dissolved/suspended organic carbon, phosphate rich zones). The act of attachment involves the microorganisms attaching, reproducing and growing outwards to form a biofilm. As the various microbial biofilms form, they interact and consortial (community) biofilms form in each of which a number of strains cooperate within a common biofilm.

Primary Void Volume Occupancy. Once a coherent consortial biofilm has formed over the surfaces, there is now a competition to occupy the void volume of the porous media. The void volume is that volume within the porous media which can be occupied by water in a saturated condition. In this initial competition between the various consortial biofilms, the volume of the biofilms expands rapidly to reach to 10 to 60% occupancy of the void volume. Transmissivity of water through the formation may be severely impaired at this time but the void volume occupancy reduces again often rapidly as the biofilms now stabilize.

Primary Stabilization. The biofilms now stabilize with a very small void volume occupancy. Transmissivity returns to pre-attachment levels and the production capacity stabilizes. At this time the well is usually considered to have been developed and goes on-line as a fully functional unit.

Secondary Void Volume Occupancy. There is now a tri-phasic pulse- like growth in the biofilms when the void volume is again gradually re-occupied by the expanding biofilms. The three phases in the cyclic growth are: (1) volume acquisition; (2) surface sloughing; and (3) restabilization. At the end of each cycle, the void volume occupancy increases impairing the transmissivity of water through the biofouling of the porous media. These cycles can cause fluctuations in the pump rate when pumping is done to a fixed draw down and the well is being operated at close to full capacity. During this phase of growth, much of the nutrients are being retained within the biofilms along with any other bioaccumulatable recalcitrant chemicals (e.g., iron, manganese). The product (postdiluvial) water coming from the well will show lower chemical concentrations due to this phenomenon except during the sloughing stage of the cycle. At this time the chemical and particulate loading in the product water may increase dramatically causing the water quality to periodically degenerate. Clog formation shifts during this phase to an encrustation or dense amorphous slime formation.

Clogging. Loss of available void volume and the growth of a clog formation leads eventually to a significant loss in the capacity of the well. The loss in transmissivity through the biofouled media (clog) restricts the amount of water reaching the well. In general practise this loss in capacity may be observed as the production falls to between 50 and 80% of the original capacity as a result of the clogging. There are two factors which influence this loss in capacity. First, there is the loss in void volume capacity to allow water flow. Second, as the clog forms within a porous medium, some zones within the formation may be isolated by a localised clogging. This can reach the state in which some of the water becomes "locked" within an encircling clog formation. This severely reduces the transmissivity of water towards the well and can cause radical reductions in the production capacity of the well.

Total Clogging. The extensive development of clog formations maturing towards dense amorphous or crystalline forms together with the "locking" of water within the porous media leads to a critical loss in the capacity of the infested well. This causes sudden, and often dramatic, drops in the capacity of the well to the point that it is no longer economical and/or desirable to keep the well on line. Sudden clogging may be expected to occur when the well is down to between 20 and 60% of the original capacity. When total clogging occurs, the capacity often falls suddenly and the well may even become essentially "dry" (producing water in such small quantities that its use is not practicable).

Unfortunately, many wells are not utilised to their full capacity. One result of this is that it is difficult for the operator of the well to appreciate that a clogging is occurring until the loss in capacity becomes greater than the production demanded of the well. By then the well may be so severely clogged that rehabilitation of the well becomes much more challenging to achieve. In the next section, emphasis will be directed at the various parameters which can be used to determine the degree of clogging which has occurred and the site and extent of the clog(s) which may be present.

Critical Indicators Reflecting the Stages of Biofouling

As the need to maintain a functioning water well of any type becomes more critical, there is going to have to be:
(1) an appreciation of the inevitability of some level of biofouling;
and (2) an understanding that the product water from the well will not necessarily reflect the risk or level of biofouling.
The challenge is therefore to develop a system which would make allowances for these uncertainties in the prediction and monitoring of the clogging risk index. Indicators include chemical, physical and biological parameters which can be used to determine the theoretical position of the clog formations within the porous media surrounding the well as risk.

Chemical Indicators Reflecting Biofouling

Since it is taken as almost certain that the water sample being subjected to analysis and interpretation is product water sampled from the well water column or downstream, the comments below relate to the determination of the clogging risk index based upon the data so gathered.
The classical techniques of determining the Biological Oxygen Demand as a measure of the (downstream) requirements for oxygen that are likely to be required are not so relevant when attempting to determine (upstream) biological activity potentials. The irony is that the analyses will tend, in the early stages of clogging, to indicate that the product water is of better quality than it really is (ie, prior to passaging through the clogging zones around the well). Chemically, the clogging becomes assessable once the clog enters the tri-phasic cycling in which the biofilms pass through growth, sloughing and stabilization phases in a repeatable manner. One effect of this is that the water will periodically contain sloughing elements from the biofilm which will materially change the chemistry of the water. This would result in the water possessing a greater variability in the targeted chemical parameters which are associated with the sloughing. Parameters likely to be included in such variability are:

Total Organic Carbon
Total Phosphorus
Total Iron
Total Manganese

During the stabilization and growth phases, the parameters will be relatively stable in concentration. Once sloughing commences, all of these parameters would increase in the water to a variable extent. Much of the increase in the chemical parameter concentration would be due to the increases in particulate loading (inputs from the sloughing of the clog) rather than in the dissolved fraction. This increase may be ascertained by filtering the water through a 0.45 micron membrane filter by gravity flow (without suction). For a given total concentration of a chemical (Ct), the dissolved fraction (Cd) would pass through the filter while the particulate fraction (Cp) would be retained on the filter. Given that the chemical may be differentiated into these two fractions:
Ct = Cd + Cp

it may be expected that in a case where sloughing contributes only to the Cp fraction, the following characteristics may be expected to occur:
     Cd would remain relatively constant,
     Cp  would increase
relative to the baseline data obtained when the clog was in the stabilization and growth phases of the cycle. Unfortunately it is not practicable to be able to monitor and project the periodicity of the triphasic cycle. In laboratory and field experiences, this cycle has been seen to occur in periods ranging from days to weeks in length. This makes the use of chemical data difficult to exploit in the prediction of a clogging event at this stage. Unfortunately, the occurrence of unusually high chemical data on some occasions may be considered by the interpreter to be anomalous events and their true role as early indicators of a clogging can therefore go overlooked. Additionally, very often the chemistry of a water sample is restricted to only the filtered sample which is essentially the Cd which may not shift much during the early phases of clog formation. It is not so common to have samples subjected to the dual analysis for the Ct and Cd fractions (in which the Cp fraction is obtained by deduction); or the Cd and the Cf fractions wherein the Ct value is obtained by addition. Perhaps the most convenient manner to determine the likelihood of a clogging event is to examine the ratio of Cp : Cd as the particulate fraction (Pf) which would shift when sloughing was occurring:
Pf = Cp / Cd

It may be expected that the Pf would remain relatively stable during the stabilisation and growth phases but would increase during the sloughing phase. Aberrant high Pf values may then be extrapolated as indicating that a sloughing event was in progress. For example, if weekly determinations were made for the Pf for organic carbon and the following results were obtained:
     week 1    -    0.45           week 2    -    0.42
     week 3    -    0.76           week 4    -    0.39
     week 5    -    0.46           week 6    -    0.84
     week 7    -    0.38           week 8    -    0.41
     week 9    -    0.91           week 10   -    0.44
Every third week, an increase in the Pf value from between 0.38 and 0.46 can be observed going up to the range of between 0.76 and 0.91. This would indicate that at those times of elevated Pf there was a greater amount of particulate-bound organic carbon which could be associated with a greater amount of particulate-bound organics in those samples. Such an event may be indicative of the sloughing of the clog formations. After such an event, it may be noted that when the clog stabilizes again, the drawdown may become reduced for a given production rate since the water flow improves.
It is currently not common for water samples to be taken and analyzed for the Pf values on a frequent (e.g., weekly) basis in order to determine the relative state of a clog formation. However, as the value of groundwater recovery and injection systems rises, there will be a greater need to implement a preventative maintenance practice which would help to ensure a managed longevity. It will become particularly important to minimize the costs of management and replacement as society becomes more environmentally concerned with maximizing the operating life span and minimizing the costs of operation.
Once the clog formation passes beyond the triphasic stage of the growth cycle towards a full maturation and clogging, the chemical parameters now degenerate with permanent increases in the Pf values as the sloughing particulate mass increases and carries with it a greater mass of bioaccumulated chemicals. As the clog matures, the particulate material may become denser and contain a higher and higher concentration of iron and manganese while the relative organic carbon, nitrogen and phosphorus loadings decrease. It is not uncommon for the sloughing particulates from a total clog to contain up to 10,000 to 20,000 ppm (dry weight) of iron in the ferric form. Once this has happened, there is a high probability of relative total clogging occurring at some points in the biofouled zones in and around the well. This has now almost totally prevented flow into the well. "Locked" water encircled by clog formations may also be found in zones within the infested porous media. Treatment now becomes much more challenging due to the occlusive nature of the matured clogs. From the chemical standpoint, instability in the selected parameters coupled to a general but erratic rises particularly in the suspended solids (particles) can form a warning system that the system is becoming badly clogged and likely to fail dramatically.

Physical Factors Influenced by Biofouling

From the previous section, it can be seen that one factor which can be used as a "marker" for biofouling and clog formation is the development of a higher and higher particulate loading. This is most easily determined as the Total Suspended Solids (TSS) and is usually measured in ppm. As the biofilms in the clogs slough so higher TSS values may be recorded in the water samples. One of the most satisfactory ways to measure TSS is by the use of laser driven particle sizing and enumeration. This technique allows the size of each particle to be determined and the volume computed together with an enumeration of the total numbers of particles. To do this, a pulsed laser light is directed through the water sample and the interference in the regularity of the pulses are used to determine the particle characteristics. From the data obtained, the laser driven particle counting is able to generate the following items of data:
     Mean particle size (microns)
     Standard Deviation in particle size
     Percentage Volume Distribution in the various allocated sizes
     Combined Surface Area
     Wet Weight (by predetermined density allocation)
This information can overwhelm the user unless a sense of the relative importance of the data can be established. Below is given some of the uses that these different data items can be put to in order to assess the presence and status of a clog formation. TSS, all of the particles that interfere with the laser beam are recorded. These range in size from less than half a micron (less than most vegetative bacterial cells) to greater than 100 microns. When there is sloughing event from a biofouled zone, the TSS will automatically increase as the particles formed by the sloughing enter the water. These particles may vary in concentration as the water moves through different formations and "flushes" away the sloughed material. When a well is subjected to a continuous pumping after a down time, very often there is an initially high TSS as the sloughed biofilms and clog material is pumped from the well. Once this has happened, the water may stabilize with a relatively low TSS value. There are techniques to use this phenomenon to measure the extent of clogging around a biofouled well. These are known as the Biofouling Assessment Quality Control (BAQC) program and involves the controlled pumping of a well in which the clog formation has been stressed to encourage sloughing.
One recently observed event which possibly can be used to differentiate a clogging well from a relatively uninfested (or newly rehabilitated) well is the nature of the bacteria infesting the water column itself. In a clogging situation, it has been frequently noticed that the TSS is not even down the water column but it forms into a series of stratified zones where different microorganisms have collected. This would mean that a series of TSS readings down such a borehole would show changes as the samples are taken from various points down the strata. In a relatively pristine well, the TSS remains homogenous down the column and stratifications are not recorded. It has been proposed that this may form a relatively simple method to determine if a well is sufficiently clogged to warrant further investigation. If the well borehole is stratified (TSS or microbial populations) then further investigations would be warranted to establish a rehabilitation/preventative maintenance program.
Mean Particle Size (microns) give the mean diameter of all the recordable particles and forms a reference point for the possible origin of the suspended particles. From the experiences to-date, the mean size can be extrapolated to the following possible conclusions:

<0.5 microns, very small particles may involve some clays, colloids and ultramicrobacteria.
0.5 - 2.0 microns, small particles may contain some bacteria but also clays and colloids may be present or dominate.
2.0 - 4.0 microns, clays and silts may be present but also some bacteria, determine whether there are some particles in the diameter range of 8 to 32 microns which would support the hypothesis of a significant bacterial population.
4.0 to 8.0 microns, suspended particles include a significant bacterial population.
8.0 to 32.0 microns, particles are large and represent sloughing elements from biofilms and/or maturing clog formations.
>32 microns, massive sloughing and break-up is occurring in matured clog formation possibly with relatively low bacterial numbers. There may also be significant silting occurring.

In general as the particle sizes gets larger on average, there is a greater likelihood that mature clogs are sloughing into the water. These particles are relatively dense and may settle quickly in static water.

Percentage Volume Distribution to the allocated sizes. On some occasions, the suspended particles may be found to have relatively specific sizes and a large percentage of the TSS may be seen to have been collected into relatively few allocated sizes. These sizes are referred to as "bins" each of which will include a relatively narrow range of particle sizes. When bacteria are present in a water sample, they may in fact be of relatively similar size and so the percentage volume distribution will be concentrated within those allocated (bin) sizes. Commonly, bacterially dominated particles within closely related particle diameters. For example strains of Pseudomonas species may commonly be found in bins ranging from 8 to 16 microns with one or two of those bins showing the greatest number of particles. Filamentous, stalked and sheathed bacteria tend to rotate in the water being examined with the result that anomalous readings are often found in the bins from 14 to 32 microns as a result of the laser beam detecting the rod-shaped cells and stalks obliquely in the light path and ascribing a diameter which more resembles the length of the rod, sheath or stalk rather than its diameter. When examining a bar graph of the distribution of the volume (or mean cell diameters) it is common to notice a "spiking" where some bins record a number of particles while the bins on either side show none detected. This "castellation" is very typical particularly for the stalks of the IRB Gallionella where these spikes may be seen 3 or 4 microns apart over the range from 14 to 32 microns.

Surface Area and Wet Weight (by predetermined density allocation). The surface area and wet weight of the TSS can be computed by most laser driven particle sizes. This allows the user to obtain an understanding of the degree of interaction that may be expected between the suspended particles and the water (surface area interface) and also the percentage incumbency of microorganisms within the suspended material through relating viable counts to the wet weight of the suspensions. This latter feature can be useful when appraising the potential involvement of suspended microbe- containing particles in remediation functions (i.e., the greater the density of microorganisms in the particles, the greater the likelihood of biological activity occurring).

Temperature is a major factor thought to influence the rate of biofouling. Most microorganisms grow within relatively narrow ranges of temperature and this is reflected in the rates of clogging generally experienced in the field. The relationship between clogging and temperature is difficult to universally establish but common guidelines are as follows:
     < 4oC -   Very slow clogging by limited range of psychrotrophs
     5 - 12oC - Slow clogging by broad range of psychrotrophs
     13 - 18oC - Clogging by a mixed psychrotroph/mesotrophic flora
     19 - 28oC - Clogging can be rapid caused by mesotrophic flora
     29 - 39oC - Rapid clogging can occur
     40 - 46oC - Clogging may be rapid with narrow spectrum flora
     47 - 75oC - Clogging generated by narrow thermotrophic flora
     >75oC -   Clogging generated by limited range of thermotrophs.
Note that psychrotrophs flourish at <15oC, mesotrophs within the range of 15 and 45oC, while the thermotrophs function within a 20 oC range at temperatures of >45oC. The temperature of the water from the well therefore can be used to obtain a general understanding of the likely rate of clogging.
Changes in water temperature being pumped from a well may also be indicative of a clogging process developing. If the clogging process is changing the origin of water flowing into the well, the temperatures of the product water may change over the time of pumping. If the clogging causes water to be redirected into the well from shallower recharge zones above the well, the temperatures of the product water may reflect the seasonal changes experienced in that water. Where water is redirected by clogging to the well from deeper groundwater strata, the temperatures may shift upwards or downwards depending upon the temperatures of the groundwaters in those formations.
There is some evidence that microbial activities associated with active biofouling including clogging may also generate heat and cause registerable changes in the water temperature. Researches are currently underway to determine the significance of this event to the diagnosis and location of clogging events in and around wells.

Pump Rates and Draw Down. Through all of the stages of clogging there are effects on the rate at which water can be produced from a well. Well production capacity is set as the rate at which a well can continuously produce water. This rate of production involves a draw down in the water level within the borehole to a position where stability is achieved. Very frequently the demand for a well is only a fraction of the production capacity of the well. The net effect of this reduced demand is to make it more difficult to record the early stages of clogging via the loss in production capacity by a well. For example, if a well had a production capacity of 1,000 litres per minute and was being pumped at a rate of 500 litres per minute, the clogging would have to reduce the flow into the well by 50% (500 litres) before there would be any stress created in the production of water from the well. What is of particular concern would be that a well already 50% clogged in terms of production capacity has a high probability of becoming totally clogged. Unless a well has been operated at close to production capacity, the use of draw-down and the rate of pumping may not be taken as being directly indicative of the rate of clogging.
In the "Armstrong" scenario, a well was pumped to a standard draw down which controlled the production of the well. A cyclic fluctuation in the production from the well was noted which reflected the triphasic stage of clogging which was occurring around the well at that time. Daily production from this well was noted to shift upwards (facilitated flow) and downwards (restricted flow) by 2 to 5% of the mean flow rates. These shifts in flow were due to the expansion, sloughing and stabilization of the developing clog and functioned over an eleven day cycle in this case. Other laboratory and field experiences have revealed similar harmonic occurrences based on cycles lasting between 9 and 120 days with many of the laboratory clogging microcosm experiments functioning in cycles of from 28 to 35 days. This phenomenon may also be observable by monitoring the draw down to a standard rate of pumping. The draw-down would not be so great during the facilitated flow phase and would be greater than the average during the restricted flow. Such phenomena cannot however be easily recorded when the well is not operating at close to production capacity (e.g., >80%)
Redox. It has been a common experience in both field and laboratory studies to find the bulk of the microbial biomass forming at the fringes where the oxygen concentrations are very low (i.e., barely oxidative) rather than in the deeper parts of the groundwater systems where there is no oxygen and the conditions are often reductive. The nature of the reductive and oxidative status of groundwater can be determined as the redox potential which is measured in millivolts. Most aerobic microorganisms function well over the redox range of +50 to +200 and become inhibited over the range from -50 to +50. This zone from -50 to +150 millivolts is sometimes referred to as the "redox front". The redox potential for water sampled from a borehole can give an indication of the likely position of a clogging formation within and/or around a borehole. For example, if the redox potential from the borehole sample the following possible conclusions could be made (redox in millivolts followed by comment):
          > +150     -   Clog likely to be deep set in porous media
          +50 - +149 -   Clog likely to be shallow set in media
          -50 - +50  -   Clog likely in borehole, pack and pump
          < -50      -   Clog likely downstream from well
Unfortunately, in a stratified biofouling within the borehole, the redox front may shift dramatically down the water column from (for example) +150 to -20 millivolts. In such a circumstance the position from which the sample for measurement was taken will critically influence the data that is obtained. Interpretation of the redox potential therefore needs to be carefully considered in the light of the possible interference factors that may effect the value of the data.

Direct Visual Inspection. While it may not be practicable to scuba dive down many wells, the use of the T.V. down hole camera to log the conditions of the casing and screen within a borehole is becoming a very popular and necessary method for logging a well. There are four major forms in which clogging may be "viewed" down the borehole but it must be remembered that the only clogging that becomes easily visible is that within the water, growing over the casing and screen and within the slots and immediate porous media behind the screens. Absence of any visible evidence does not mean that no clogging is occurring. The forms of clogging are:
Freefloating. Clouded water and the presence of floating particles usually with very ill-defined edges indicate the probability that there is a considerable amount of microbial growth present within the water column itself. Sometimes this growth can become so dense as to black out the light used to illuminate the casing and screen.
Mucoid Tubercles. When the clog is forming just behind the well screen and is growing rapidly, mucoid tubercles (resembling in shape "cabbages" or "cauliflowers") grow into the well through the screen. These growths are often very fragile and break apart very easily when knocked with the camera housing or lights. The water can become so cloudy that visibility is completely lost. These slimy ingrowths into the borehole signify the presence of a clog formation which is probably growing for the most part outside of the borehole in the porous media in close proximity to the well.
Hardened Plates. These may be seen as plates of rust hanging down the casing and/or the screens. Unlike the tubercles, the plates "rusticles" are relatively rigid and can resist minor knocks and abrasions which may be caused by the equipment installed or admitted to the borehole. Such plate-like growths indicate that a matured encrustation is present and the clogging process may be well advanced. These plates represent the inner edges of a matured clog formation which may extend for some considerable distance back into the porous media around the borehole.
Covert Clogging. The first impression when there are no signs of growth in or around the borehole is to assume that there is no clogging taking place. While this may be the case, there remains the possibility that there is a covert form of clogging forming beyond the field of vision. That is to say, it is growing within the porous media surrounding the borehole. A relatively high (oxidative) redox value (i.e., >+ 50 millivolts) for the water in the well and a knowledge that there is likely to be nutrients reaching the well in sufficient quantities to stimulate biofouling can both be taken as the positive indicators of the probability of clogging occurring within the formation.

Biological Factors influenced by Biofouling

It is generally believed that the majority of the biological factors affecting biofouling and clog formation are bacterial in origin. In the deeper fouled zones where the redox front resides and beyond, this would certainly be the case. However, in oxidative environments (redox > +50) there may also be significant populations of protozoa feeding on the bacterial growths. Occasionally micro-algae may also be present. These microplants are normally considered to be plants and to obtain their energy from the photosynthesis of (sun)light. Many have also been found to be able to survive and even flourish in the dark using organic sources of carbon. Molds (another major group of microorganisms) can also be recovered from well waters which have been recharged from unsaturated zones within the aquifer. When this happens the groundwater may carry very high spore populations. Spores may bias the attempts to enumerate the microorganisms present in the water by overgrowing the agar plates. Earthy-musty odors are sometimes associated with the presence of these organisms and, in particular, a group of bacteria called the Streptomyces.
Enumeration of the microbial populations within a biofouled and clogging water well is made particularly difficult for a number of reasons. These include:
Attached habitat. Most of the microorganisms present in a biofouled well tend to grow attached to surfaces either as loose slimes or maturing clog formations. The organism in suspension in the water itself tend to aggregate into suspended "colloidal" masses which may contain a mixture of different strains functioning in a consortial manner. Only a relatively few bacteria will grow ("swim") independently within the water column. This creates a significant problem when attempting to determine the scale of a microbial population within a biofouling water well. It is quite possible for water drawn from a heavily biofouled water well to contain no detectable microorganisms simply because all the organisms are attached and not actually suspended in the water as such. Even if there were microorganisms in the water, the fact that they are commonly in a suspended "biocolloidal" form means that enumeration becomes difficult using the classical spreadplate techniques in which the bacteria are grown up on an agar surface to form separate and distinct countable colonies.
Oligotrophic Nutrient Regime. Oligotrophic means "slight" growth due mainly to a shortage of suitable nutrients. Most of the microorganisms are in a state of nutrient starvation and relatively traumatized. When such microorganisms are taken in a water sample, there is often a recovery period which is necessary before the organisms can begin to grow. Such traumatised organisms are often not enumerated correctly simply because the culturing (incubation) times are kept to a minimum in order to "turn around" the samples in a reasonable time for the users. Better recoveries are achieved where the cultural conditions are changed more slowly thus allowing the organisms time to adapt to the richer and more nurturing nutritional regimes that are created in the laboratory.
Erratic Population Recording. There are so many variables in the enumeration of microorganisms in the water ranging from stratification in the water column of the well, randomized and periodic sloughing from the attached slimes to unstable biocolloidal particles which sometimes break up dispersing the incumbent bacteria into the water.
Specialized Growth requirements. Many of the microorganisms within the water may require very specific environmental and cultural conditions for growth which may not necessarily be generated when the laboratory attempts to culture these organisms. Very often total counts of microorganisms in water using techniques such as the Acridine Orange Direct Count (AODC) method reveal far more microorganisms than are enumerated using the classical agar spreadplate techniques. Often three orders of magnitude higher numbers are recorded when the AODC is used rather than the standard spreadplate agar media.
Of the agar media, there are a wide variety which have been used to enumerate bacteria. Popular agar culture media include R2A (heterotrophic bacteria), Glucose Tryptone Soy (fastidious heterotrophic bacteria), WR (iron related bacteria) and potato dextrose (molds). In general, there has been a lack of consistency in the data generated using the agar plate spreadplate because of the factors discussed above. The variability in the data so obtained has led to a loss in confidence when attributing a clogging event to a microbial origin. As a result, alternative techniques have been developed in an attempt to improve the reliability of the data.
One simple technique which has recently been developed and patented is the biological activity reaction test (BARTTM) which compensates in some manner for the various shortcoming in the present techniques applied to determine microbial challenges in groundwater. The basic premise is to eliminate shock during the transfer of organisms from their natural environment to the evaluatory conditions. This is done using the concept of the Winogradsky column in which redox conditions are established from oxidative (top of the column) to reductive (bottom of the column). This is exaggerated in this test by the insertion of a floating ball to reduce the oxidation zone. Nutrients to support growth gradually diffuse up from dried pellets in the base of the tube. Counter gradients are therefore created with nutrients diffusing upwards while the oxygen diffuses downwards. Microorganism may therefore locate and grow at suitable sites along these gradients within the water sample.

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