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  1. The Science Behind BioShield

  2. Let’s consider the issue of microbiological contamination of groundwater wells, which impacts affordability, source sustainability and public health.

  3. According to the USGS, groundwater sources supply approximately 20% or 84.6 billion gallons per day of fresh water used in the United States.

  4. These precious groundwater resources must be managed and protected by implementing strategies to ensure groundwater quantity, such as well yields, and groundwater quality with respect to bacteriological impacts.

  5. Is groundwater a sustainable resource? The number one reason for loss of production and abandonment of ground water wells is Biofouling. The number one agent of biofouling is iron related bacteria.

  6. Biofouling or biological fouling is the undesirable accumulation of microorganisms on submerged structures. Individually small, accumulated biofoulers can form enormous masses. Biofouling can occur in groundwater wells where buildup can limit recovery flow rates and clog pumps and piping.

  7. A variety of bacteria are indigenous to soils and groundwater. These are mineral utilizing species of bacteria which exist in low numbers in groundwater as this is a low nutrient environment.

  8. Bacteria in nature are most frequently encountered not as free-swimming organisms but as surface-attached communities known as biofilms.7 Organisms residing within biofilm possess a number of advantages over their free-swimming or planktonic counterparts, including increased resistance to adverse environmental conditions and antibacterial agents.

  9. The ubiquity of biofilm development can cause significant problems in the areas of public health, 8, 9 medicine, 10, 11, 12 and industry. 13, 14 Accordingly, there has been a great deal of research to better understand biofilm development and to identify improved strategies for biofouling control.

  10. Earth’s Composition • 5% Earth’s crust is Iron • Almost every groundwater source contains measurable amounts of Iron

  11. “Surface waters contain an innumerable variety of organics from municipal or industrial wastewater effluents, storm water runoff, agricultural activities and natural vegetation, producing humic substances. Total organic concentrations range from 1 to 10 mg/L at the water supply intake with 7 mg/L on average.”1 “For aquatic systems the organic matter includes dissolved organic carbon (DOC) and particulate organic carbon. Groundwater systems are frequently among the most oligotrophic microbial environments that have ever been described (mean concentration from 0.1 to 0.7 mg/L).”2 However, where one measures high organic carbon in the subsurface water, this difference is attributable to microbial activities. Bacterial carbon production rates are extremely high in biofilm communities.3

  12. However, when these bacteria enter groundwater wells, they become attached to surfaces within the well, particularly areas of high velocity, and they begin to dramatically increase in number.

  13. National Secondary Drinking Water Regulations National Secondary Drinking Water Regulations (NSDWRs or secondary standards) are non-enforceable guidelines regulating contaminants that may cause cosmetic effects (such as skin or tooth discoloration) or aesthetic effects (such as taste, odor, or color) in drinking water. EPA recommends secondary standards to water systems but does not require systems to comply. However, states may choose to adopt them as enforceable standards.

  14. Impact of Pumping Biofouling caused by iron related bacteria can occur at very low levels of iron due to the impact of pumping in the well proper.

  15. This sessile, or attached, state is highly immune to biocides, enjoying from 150-3000 fold immunity. At the same time, this sessile form, which excretes a polymeric substance as well as insoluble forms of the minerals they utilize as hydroxides, begins to build up clogging structures within the well, pump and distribution piping. The water these wells produce also contains these polymeric substances (extracellular polysaccharides) and the ferric hydroxides as well as sulfate compounds which are very corrosive. In addition to the obvious problems associated with such bacteria, the slime they produce will entrain any minerals in the gravel pack as water moves into the well. This mineral encrustation can render a well useless in very short order. Are you keeping pumping records? Have you noted a decline?

  16. Steps in Biofilm Development The instant a clean pipe is filled with water, a biofilm begins to form.

  17. Step 1: Surface conditioning • The first substances associated with the surface are not bacteria but trace organics. • Almost immediately after the clean pipe surface comes into contact with water, an organic layer deposits on the water/solid interface (Mittelman 1985). These organics are said to form a "conditioning layer“ which often serve as a nutrient source for bacteria. Adsorption of organic molecules on a clean surface forms a conditioning film. (Characklis 1990)

  18. Step 2: Adhesion of ‘pioneer’ bacteria • In a pipe of flowing water, some of the planktonic (free-floating) bacteria will approach the pipe wall and become entrained within the boundary layer, where flow velocity falls to zero. • Cells begin to form structures which may permanently adhere the cell to the surface. Transport of bacteria cells to the conditioned surface. (Characklis 1990)

  19. Attachment = Resistance • The attachment of bacteria to surfaces increases resistance to biocides including chlorine from 150 to 3000 fold. • Other factors (from 2 to 10-fold) include biofilm age, encapsulation, and growth conditions. • Factors are mulitplicative. LeChevallier, Cawthon, Lee. Factors promoting survival of bacteria in chlorinated water supplies. AWW Service Co.

  20. Phenotypic plasticity The ability of bacteria to adapt to environmental changes is known as phenotypic plasticity. A good example of phenotypic plasticity is planktonic bacteria in groundwater utilizing a sessile lifestyle in groundwater wells.

  21. Step 3: Extracellular Polysaccharides ‘slime’ formation • Biofilm bacteria excrete extracellular polysaccharides, or sticky polymers, which hold the biofilm together and cement it to the pipe wall. • These polymer strands, much like the structure of a spider’s web, trap scarce nutrients and protect bacteria from biocides. • This extracellular slime acts as an ion-exchange system for trapping and concentrating trace nutrients from the overlying water. • The slime acts as a protective coating for the attached cells which mitigates the effects of biocides and other toxic substances.

  22. Step 4: Secondary Colonizers • As well as trapping nutrient molecules, the slime net also snares other types of microbial cells. • These secondary colonizers metabolize wastes from the primary colonizers as well as produce their own waste which other cells then use.

  23. Step 5: Fully Functioning Biofilm  A cooperative "consortia" of species • The mature, fully functioning biofilm is like a living tissue on the pipe surface. It is a complex, metabolically cooperative community made up of different species each living in a customized microniche. • Biofilms are even considered to have primitive circulatory systems. • Different species live in concert helping each other exploit food supplies and resist antibiotics. Toxic waste produced by one species is utilized by another. Functioning symbiotically several species of bacteria, each armed with different enzymes, can break down food supplies that no single species could digest alone. • The biofilms are permeated at all levels by a network of channels through which water, bacterial garbage, nutrients, enzymes, metabolites and oxygen travel to and fro. Gradients of chemicals and ions between microzones provide the power to shunt the substances around the biofilm." (Coghlan 1996)

  24. Biofilms grow and spread •  A biofilm can spread at its own rate by ordinary cell division and it will also periodically release new ‘pioneer’ cells to colonize downstream sections of piping. • According to Mayette (1992), "These later pioneer cells have a somewhat easier time of it than their upstream predecessors since the parent film will release wastes into the stream which may serve as either the initial organic coating for uncolonized pipe sections down stream or as nutrient substances for other cell types."

  25. Biofilm Cross Section Bacteria and other microorganisms develop cooperative colonies or "consortia" within the biofilm. An anaerobic biofilm may develop underneath the aerobic layer. (Borenstein 1994) Note: Coliforms are facultative; they can live in anaerobic or aerobic conditions.

  26. How fast does biofilm develop? • According to Mittelman (1985), the development of a mature biofilm may take several hours to several weeks, depending on the system. Pseudomonas aeruginosa is a common ‘pioneer’ bacteria and is often used in biofilm research. In one experiment (Vanhaecke 1990, see test summary pg 11), researchers found that Pseudomonas cells adhere to stainless steel, even to electropolished surfaces, within 30 seconds of exposure.

  27. Bacteria exploit every environmental niche on the planet • Bacteria exist at the top and bottom of every known food chain • They are found in: • Pack Ice • Undersea superheated vents • Low pH • High pH • Freshwater – Saltwater This attests to their adaptability and broad phenotypic plasticity.

  28. Think about it! Bacteria can easily gain entrance to the water well: Well head Soil Grout seal Rock Water Table Groundwater “aquifer”

  29. Many of man’s activities cause bacterial contamination of ground water ! Residential Landfills, dumps Gas - Oil Wells Industry Agriculture Mining

  30. Location of our water well is important

  31. Ground water in its natural state is not always free from problem causing bacteria

  32. Microphotographs of bacteria that precipitate iron and manganese • Gallionella ferruginea • Leptothrix cholodnii • Thiobacillus • Siderococcus

  33. Why be concerned about iron related bacteria in groundwater wells? • Biofouling caused by iron related bacteria destroys groundwater well and distributions assets. • Opportunistic and pathogenic bacteria colonize biofilm.

  34. Distribution Asset Destruction Pipe wall Biofouling - Example 4 inch pipe 3/16 inch thick = 12% reduced flow area 3/8 inch thick = 36% reduced flow area 9/16 inch thick = 51% reduced flow area 3/4 inch thick = 64% reduced flow area 1 inch thick = 75% reduced flow area

  35. Effects of IRB • Clogged and corroded piping with rusty sludge (ferric hydroxides) • Increased chances of sulfate reducing bacteria infestation – MIC (microbe-induced corrosion) • Unpleasant odors and taste • Increased organic content in water favoring the multiplication of other bacteria • Severe damage to pumping equipment • Seriously impacts water treatment • Reduces distribution efficiency • Severe blockages in gravel pack, well and screens

  36. Chronic bio-fouling caused by Iron bacteria Many wells have been abandoned because of biofouling.

  37. Water flow area virtually eliminated

  38. Emerging Microbiological Issues Iron Related Bacteria (Iron, Sulfate, Manganese Utilizing Species) Galionella Ferruginea Sphaerotilus Leptohrix E-Coli Can encourage pathogen growth

  39. Consortia bacteria in a biofilm • IRB • SRB • Coliform Bacteria • Opportunistic bacteria - Pseudomonas aeruginosa • Pathogenic Bacteria - H. pylori

  40. List of Contaminants & their MCLs

  41. Drinking Water Contaminant Candidate List 2 On February 23, 2005, EPA announced the second Drinking Water Contaminant Candidate List (CCL) and our efforts to expand and strengthen the underlying CCL listing process to be used for future CCLs. • Microbial Contaminant Candidates • Adenoviruses • Aeromonas hydrophila • Caliciviruses • Coxsackieviruses • Cyanobacteria (blue-green algae), other freshwater algae, and their toxins • Echoviruses • Helicobacter pylori • Microsporidia (Enterocytozoon & Septata) • Mycobacterium avium intracellulare (MAC)

  42. Pathogenic Bacteria Colonize Biofilms In Western countries rates of Helicobacter pylori infection are as high as 60% by age 65. Infection with H. pylori is now recognized as a causative agent in chronic gastritis, as well as peptic and duodenal ulcer disease. In addition, infection with this organism is associated with mucosa-associated lymphoid tissue lymphoma and adenocarcinoma. Epidemiological data support transmission through a common source such as water. Recently the USEPA Office of Groundwater and Drinking Water included H. pylori in its contaminant candidate list reflecting concerns over possible waterborne transmission. Studies have shown H. pylori to be significantly more resistant to chlorine than E. coli. In the natural environment, H. pylori form a biofilm in 1 to 15 days. Biofilm bacteria were found to be 150 to 3,000 times more resistant to hypochlorous acid than similarly treated unattached microbes. Several researchers have examined the possible role of biofilms in the proposed waterborne transmission of H. pylori and have demonstrated that H. pylori are capable of forming biofilms and of persisting in mixed-species drinking water biofilms. 18

  43. Changes in human demographics There is an increasing number of “vulnerable subpopulations” in the United States such as infants, children, pregnant women, the elderly, and the immune-compromised who are particularly susceptible to infections resulting from exposure to waterborne pathogens compared to the general populace. • Immune-compromised: • Cancer, Crohn’s disease, Lupus, HIV, AIDS • Transplant – organ, bone marrow, stem cell • Rheumatoid Arthritis, diabetes • Steroid use, long term anti-acid users >2 weeks • Smokers

  44. Chlorine is not effective • Studies have shown that despite the application of continuous disinfection using chlorine biofilm rapidly develops. Biofilm can rapidly form in the presence of a 1 to 2 mg/L free chlorine residual.6 • Given that chlorine is ineffective, what can be done to remediate or eliminate biofilm and the associated health risks in groundwater?

  45. Stressing bacteria is not the total solution Stressing bacteria with certain biocides and/or physical treatments such as heat or mechanical removal is not the total solution. 1.) Bacteria have short life cycles (20 minutes) which promote a high level of adaptability to environmental changes. 2.) Microbiological structures within a biofilm protect bacteria from environmental fluctuation. 3.) According to Characklis (1990), biofilm recovers from stressing.

  46. According to Characklis (1990), biofilm recovery may be due to one or all of the following: • The remaining biofilm contains enough viable organisms that there is no lag phase in regrowth. Thus, biofilm recovery after shock chlorination is faster than initial accumulation on a clean pipe. • The residual biofilm on the surface makes it rougher than clean pipe. The roughness of the deposit may provide a stickier surface which adsorbs more microbial cells and other compounds from the water. • The chlorine preferentially removes extracellular polymers and not biofilm cells, thus leaving biofilm cells more exposed to the nutrients when chlorination ceases. • Surviving organisms rapidly create more slime (extracellular polymers) as a protective response to irritation by chlorine. • There is selection for organisms less susceptible to the sanitizing chemical. This is usually the organisms that produce excessive amounts of slime like Pseudomonas. 

  47. Shock chlorination is not the solution Shock chlorination is not the solution. Chlorine is a common disinfectant used in water systems, and is highly toxic to coliform and similar types of bacteria. IRB and SRB are more resistant to chlorine’s effects because they occur in thick layers and are protected by the slime they secrete. A standard chlorine treatment may kill the bacterial cells in the surface layer but leave the rest untouched. In the case of iron bacteria, iron dissolved in the water may absorb disinfectant before it reaches the bacteria. For all these reasons, iron and sulfate related bacteria may be able to survive a chlorine treatment that would kill other types of bacteria. Treatment levels of 800-1,000 ppm chlorine are commonly recommended for treatment of iron and sulfate related bacteria; however, these levels promote the growth of coliforms. At best, shock chlorination is minimally effective for a limited period of time.