Bio-Reactor Technology and Applications – a White Paper · TVT Bio-Reactor Technology and Applications | 6 Biofilms – the engine of bioremediation Biofilms are microbial populations

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CONTRIBUTORS: Kenneth Rainis, MS Jim Hyzy, PhD. Richard Chape, P.E. Joyce McChesney, M.D.

Environmental Treatment Solutions TVT US Corporation, Inc. PO Box 25090 Rochester, NY14625 INFORMATION: www.tvt-bio.com DIRECT: 1-585-264-1058 FAX: 1-585-385-4019 February 22, 2010 Copyright 2010 TVT-Bio

TVT Bio-Reactor Technology and Applications | 2

Table of Contents About TVT-Bio® …………………………………………………………...... 3 Company Patents …………………………………………………………….. 4 The TVT Process of Bioremediation ……………………………………….. 5 What is Bioremediation What is a Bio-Reactor? Biofilms – the engine of bioremediation TVTs Bioremediation Process Aerobic and Anaerobic Treatment – combined Effeciency Process Applications

Case Histories ……………………………………………………………….. 10 Winery Application Cheese / Dairy Manufacturing Dairy Farming Steel Plant Food Processing (Canning)

Current Research: Hypersaline Remediation ………………………….. 15

Testimonials ………………………………………………………………….. 16 Selected References ………………………………………………………… 17 Appendix (Company Patents) ……………………………………………… 18 Glossary ………………………………………………………………………. 20 TVT Bio-reactor ………………………………….…………………….......... 28


About TVT-Bio® Headquarters: Rochester, New York PRESIDENT Volker Hausin [email protected]

Incorporated in 1997 to provide a unique solution to industrial, municipal, and agricultural wastewater treatment compliance requirements, the company’s patented process employs an extremely efficient aerobic fixed biofilm bioremediation treatment strategy that is application-specific, cost-efficient, and tailored to a wide variety of effluent waste stream types. TVT-Bio® equipment has proven successful in a variety of industrial applications in both equalization and activated sludge basins. Unique aeration process efficiencies, combined with the bio-breeding chamber, reduce the expense of continuous microbial insertions required in many biological systems. The combination process also reduces the need for a large footprint, additional aeration equipment, and infrastructure additions. Optional instrumentation can monitor (log or transmit) a variety of treatment parameters, including: dissolved oxygen, pH, total dissolved solids, temperature & conductivity.


Company Patents See Appendix for abstracts

The Company’s patents effectively establish cascading technology support around overcoming process limitations associated with poor settling of sludge in conventional activated sludge (CAS) processes. The TVT-Bio-Reactor permits higher mixed liquor suspended solids (MLSS) concentration than CAS systems, which are limited by sludge settling. The patents address a unique methodology that combines velocity-managed and oxygen-rich linear flow streams and log-phase bio-augmented fixed biofilm to increase native microbiological metabolic efficiencies in bioremediation of organic waste. United States Patent 60/909,954 FIXED-FILM BIOPROCESS FOR REMOVING CARBON COMPOUNDS IN OIL AND GAS DRILLING SLUDGE United States Patent 60/941,272 VERTICAL LAND-BASED BIOREACTOR United States Patent 6,022,476 WATER TREATMENT PROCESS United States Patent 6,231,766 WATER TREATMENT PROCESS United States Patent 6,821,426 PROCESS FOR TREATING A BODY OF WATER United States Patent 7,101,483 WATER TREATMENT PROCESS


The TVT Process of Bioremediation In totality, the Company’s patents encompass an efficiency improvement in activated sludge treatment bio-reactor technology having wide industrial wastewater treatment applications in a wide variety of market segments including: government, industry, and agriculture. Examples include:

- Winery - Steel Mill - Dairy Farming - Cheese / Dairy Processing - Food Processing - Gas, Oil Drilling - Oil Sands

What is Bioremediation? Bioremediation is any process that uses microorganisms, or their metabolic products, to return the natural environment, altered by contaminants, to its original condition. What Is a Bio-Reactor? A bio-reactor is a device or system that sustains a biofilm as part of an aerobic or anaerobic process or is involved in the cultivation of an organism or special group of organisms. There are two main bioreactor types: moving media and fixed-film. Fixed-film bioreactors grow microbes on substrates such as rocks, sand or plastic. The wastewater is spread over these substrates, allowing the wastewater to flow past the film of microorganisms fixed to the substrate. This provides an extremely efficient way of allowing microbes an opportunity to feed on dissolved nutrients1. Trickling filters, rotating biological contactors, sand filters and aquarium filters are examples of fixed film systems.

TVT fixed-film bioreactors are designed to achieve a unique laminar flow efficiency – moving contaminants to an ever-growing biofilm2 to achieve maximum contact frequency and microbial productivity.

1 The patented design efficiency of TVT’s bioreactor provides for microbial growth of 109 within the lagoon containment applications conditions as compared to the normal cell density presence of 104 . 2 As a biofilm grows its efficiency starts to deteriorate—its increasing thickness starts to put up barriers to nutrient and gas exchange. The key to biofilm productivity is to provide for a constant renewal of the film so that only actively growing portions are in contact with nutrient-rich waste streams. TVT’’s patented technology is key to achieving this process application.


Biofilms – the engine of bioremediation Biofilms are microbial populations that adhere to surfaces. Examples of these fixed biofilms include—dental plaques, green coatings on wet surfaces, coatings on the glass walls of aquariums and aquarium gravel filters. Biofilms vary in thickness, from microns to centimeters—each a small and unique ecosystem composed of bacteria, microfungi, alage, protozoa, and even microinvertebrates3 Biofilms are held together and protected by secreted chemical matrix that protects component member cells. In a biofilm, the microbial community thrives on the energy they acquire from carrying out transformations of various “food” sources brought to it in the surrounding fluid environment. The life of a biofilm starts with free-floating cells4. In order for a floating cell to attach to a surface, it must first interact with the surface. Surfaces immersed in a solution usually acquire a surface charge that attracts and concentrates inorganic and organic molecules. The concentration of this “molecular strew” can provide a nutritious zone for bacteria and other microbes compared to the “ocean” of liquid that surrounds it. Several parameters affect how quickly biofilms form and mature, including surface, cellular, and environmental factors5. Although a biofilm can (or will) form on any surface, plastics are the most efficient biofilm surface generators. This is believed to be due to the material’s electrical charge characteristics and its hydrophobic characteristics. Specially-designed bioreactor substrates are called media.

3 Single species biofilms are used to create various industrial materials such as 2,3-butanediol from whey.

4 Certain cells have exclusive energy appetites. For example, certain Pseudomonas bacteria species will devour hydrocarbon chains in oils. Other bacteria (Kocuria sp., Brevibacterium linens and Staphylococcus sciuri) are partial to whey. The list of microbes and their food preferences is almost unlimited! 5 Environmental factors such as temperature and pH can have an effect on biofilm formation. Depending upon the microbe, high temperatures increase the rate of cell growth and surface adhesion. pH values that are less than 4 units or greater than 9 units can drastically reduce microbial diversity and thus affect biofilm formation.


TVT’s Bioremediation Process: The fixed film TVT Bio-Reactor system incorporates three complementary waste treatment modalities: (1) laminar flow (velocity management) of aerated wastewater, (2) substrate composition and design, and (3) bio-augmentation6. Typically, aeration mixing is provided for by high-horse-power splashers and aerators above the biofilm matrix. Laminar-Flow Velocity Management The TVT Bioreactor utilizes a submerged laminar flow coupled with air injection to achieve continuous delivery of nutrient-laden wastewater to a growing biofilm. The patented aeration system allows maximum diffusion of oxygen at the water-biofilm interface. BioFilm Substrate Specially-designed biofilm substrate7 having an ionic coating which permits rapid adherence of microbes to facilitate film-building. Unique, patented geometric design allows for shedding of thickening layers and allows for continuous biofilm renewal under laminar flow conditions. Bioaugmentation Besides creating unique physical conditions of oxygenated laminar flow over a fixed substrate, bio-augmentation plays a critical part in bioremediation success. TVT has two sources of application-specific microbiology: (1) a repository of free-living, proprietary, aerobic application-specific microbial strains and (2) recovered custom activated sludge process bio-film microbial communities. TVTs proprietary microbial isolates include: extreme halophiles for bio-processing high salt (chloride) wastewater (brine), VOC-degrading bacteria, yeasts, and fungi, denitrifiers (facultative heterotrophic bacteria8), and polyphosphate-accumulating organisms (PAO9).

6 Introduction of a group of natural microbial strains.

7 The plastic biofilm substrate has 32 ft2/ft3 of round surface area when new. As biofilm builds up on the matrix substrate, the round surfaces of the biofilm in contact with the wastewater increase geometrically with the thickness of the biofilm. This enlarged surface can expand the effective surface of the matrix substrate to 60 to 90 ft2/ft3. As the mass of the biofilm increases, it will eventually slough off the substrate matrix elements, aided by the laminar flow streams inside the bioreactor.

8 Pseudomonas, Bacillus and Achromobacter that convert nitrate in wastewater to nitrogen gas.

9 Specific bacteria, called polyphosphate accumulating organisms (PAOs), accumulate large quantities of phosphorus within their cells (up to 20% of their mass). When the biomass enriched in these bacteria is separated from the treated water, these biosolids have a high fertilizer value.


Aerobic and Anaerobic Treatment - combined The TVT Bio-Reactor can be configured to run both aerobic and anaerobic biofilm bioremediation working in synergy. Such dual remediation strategies are effective in promoting anaerobic reductive dehalogenation10 and aerobic co-metabolic biodegradation,11 simultaneously, in a single biological reactor. Efficiency The biochemical oxygen demand (BOD12) test has been used widely by regulatory agencies to gauge overall treatment plant efficiencies. The traditional BOD measurement of the plant influent and the final effluent gives the most common measure of treatment plant efficiency. The BOD of wastewater is a common indicator of the fraction of organic matter that may be degraded by microbial action. The test is related to the oxygen that would be required to stabilize the waste after discharging to a receiving body of water.

This unique concept of combining velocity-managed and oxygen enriched wastewater to a log-phase-growth fixed bio-augmented biofilm can increase operational efficiencies up to 73%.

Process Applications: - Improve efficiency and capacity of municipal wastewater (conventional activated sludge - CAS) treatment systems13

- Controlling nitrification14—conversion of ammonia to nitrates. - Phosphorous removal utilizing PAOs - Increasing dissolved oxygen to reduce biological oxygen demand (BOD). - Controlling of fats, oils, and grease (FOG).

10 The biologically-mediated replacement of chlorine (as chloride) on a chlorinated organic compound. 11 Co-metabolism is an approach to biological degradation of hazardous solvents. Using methane as the primary energy source, some microbes release enzymes that degrade the chlorinated solvents. 12 BOD measures the rate of oxygen uptake by micro-organisms in a sample of water at a temperature of 20°C and over an elapsed period of five days in the dark. Most pristine rivers will have a BOD below 1 mg/L. Moderately polluted rivers may have a BOD value in the range of 2 to 8 mg/L. Municipal sewage that is efficiently treated by a three-stage process would have a value of about 20 mg/L or less. Untreated sewage varies, but averages around 600 mg/L.

13 The TVT Bio-Reactor effectively overcomes the limitations associated with poor settling of sludge in conventional activated sludge (CAS) processes. The TVT-Bio-Reactor permits higher mixed liquor suspended solids (MLSS) concentration than CAS systems, which are limited by sludge settling. The process is typically operated at MLSS in the range of 8,000–12,000 mg/L, while CAS are operated in the range of 2,000–3,000 mg/L. The elevated biomass concentration allows for very effective removal of both soluble and particulate biodegradable materials at higher loading rates. Thus complete nitrification even in extremely cold weather is feasible. Placing the TVT Bio-Reactor in a lagoon provides increased effluent polishing by invertebrates such as Daphnia and rotifer species by removing fine particulates. 14 Nitrification itself is a two-step aerobic process, each step facilitated by a different type of bacteria. The oxidation of ammonia (NH3) to nitrite (NO2

−) is most often facilitated by Nitrosomonas spp. Nitrite oxidation to nitrate (NO3

−), facilitated by Nitrospira spp.


- Increased oxygen transfer increasing microbial processing efficiency. - Aerobic15 digestion. - Anaerobic16 digestion. - VOC17 / Oil Sands18 process remediation. - Chloride reduction (brine conditions > 5% salt content) - Phenolics and chloronated hydrocarbons19 - Increasing biodiversity (through bioaugmentation) of mixed liquid suspended solids (MLSS)20 - Groundwater treatment. - Acid rock drainage (ARD21) from mining operations.Its versatile design (aerobic / anaerobic configuration) allows the TVT Bio-Reactor to efficiently treat ammonia, nitrites, nitrates, and phosphates by both aerobic and anaerobic microbiological pathways.

Process Benefits:

- Reduction in treatment time. - Odor control (usually within 1 month of startup). - Sludge reductions (30-50%). - Lower operating costs (reductions of electricity and chemical use). - Regulatory compliance regarding discharge limits.

15 Aerobic digestion is a bacterial process occurring in the presence of oxygen. Under aerobic conditions, bacteria rapidly consume organic matter and convert it into carbon dioxide. 16 Anaerobic digestion is a bacterial process that is carried out in the absence of oxygen. Fermentation is an example of an anaerobic process. 17 VOC – volatile organic compounds. 18 Oil sands are naturally occurring mixtures of sand or clay, water and an extremely dense and viscous form of petroleum called bitumen. 19 E.g. trichloroethylene; vinyl chloride. Dehalococcoides ethenogenes is the only known bacteria, grown in consortia with other bacteria that can fully degrade PCE to ethane under anaerobic conditions. 20 Most sewage treatment plants (STPs) tend to fail due to one or more of the following: (1) inadequate population of microorganisms measured as MLSS, (2) inadequate aeration, and (3) loading above design capacity. Microbial genera employed by TVT include: B. licheniformis, B. thurengensis, B. polymyxa, B. sterothemophilus, Penicillium sp., Aspergillus sp., Flavobacterium, Arthrobacter, Pseudomonas, Streptomyces, Saccaromyces, Triphoderma, etc.). Metazoan growth status, and the breeding of metazoans accelerates flocculation capability of bacteria and improves the performance of the sludge sedimentation.

21 Sulfate-reducing bacteria are nurtured to generate sulfides that scavenge dissolved metals to form metal sulfide precipitates.


CASE HISTORIES Case History: Winery Application Background A winery in Upstate New York needed to increase mixed liquor treatment capacity. The winery wastewater treatment plant consists of a series of concrete lined circular ponds. A Head works followed by the equalization/mixing basin, followed by two long-retention, activated sludge ponds, followed by two clarifiers, and finally a sand filter. The daily inflow is 32,000 GPD with peak loads at crushing time up to 90,000 GPD. Pond 1, the equalization/ mixing pond has a diameter of 72’, pond 2, the first activated sludge pond has also a diameter of 72’, and pond 3, also activated sludge, has a diameter of 90’. The activated sludge empties into both clarifiers and from there is passed through the sand filter to be finally discharged into a trout stream. Application Task Increase liquor treatment capacity; accommodate various (changing) manufacturing processes with daily influent of 32,000 GPD (gallons per day). Balance microbiology so that process BOD loading can be accommodated. Methodology Installation of the TVT-BIO 32 System in the 96,000 gal. primary treatment lagoon. Impact: (following an operation period of ten months)

� Unit installation occurred within 2 hours; no crane needed. � Significant reduction in odors. Greatly reduced need for additional bio-augmentation. � Increased and stabilized biofilm biodiversity on circular pond concrete walls22. � Increased liquor recirculation / contact time – 11X / 24 hr. � Decreased power consumption (30 - 40 %). Prior plant consumption was 150hp. Installation

of the TVT Bio-reactor resulted in a power utilization decrease of 20 to 60hp with a maximum reduction to 86.5hp.

� Decreased BOD loading cycles – up to 50%. � Increased flocculation; significant reduction in foaming. � COD: 1,000+ mg/L reduced to ~ 100 mg/L.LSS (2) ionadequate aeration, andf � BOD23: Treated levels ~ 3-4 mg/L. � Sludge reduction � Quick recovery to normal levels of BOD, which previously have taken ten days, take 2-3 days. � Limited flocking had previously occurred. With use of the TVT system a more developed and

constant flocculated structure appears in the mixing/equalization basin. Higher mixed liquor suspended solids (MLSS).

� Significant reduction of foaming. � Quick recovery to normal levels of BOD.

22 Ciliates, rotifers, free swimmers, amoeba, and even nematodes (before not noted in quantity). 23 Most pristine rivers will have a 5-day carbonaceous BOD below 1 mg/L. Moderately polluted rivers may have a BOD value in the range of 2 to 8 mg/L. Municipal sewage that is efficiently treated by a three-stage process would have a value of about 20 mg/L or less. Untreated sewage varies, but averages around 600 mg/L. Ref: Clair N. Sawyer, Perry L. McCarty, Gene F. Parkin (2003). Chemistry for Environmental Engineering and Science (5th ed.). New York: McGraw-Hill. ISBN 0-07-248066-1.


Case History: Cheese / Dairy Product Manufacturing Background A cheese manufacturer with a 3 million gallon earth lagoon, discharges whey24 onto an adjacent meadow. Typically the lagoon establishes an active green algae population during summer operations. During winter operations the daily whey wastewater is discharged directly into the lagoon system, which has enough capacity to retain all whey discharge until February / March. The collection tank in the factory has fluctuating BOD loadings from 3,860-10,600. The pH range in the collection tank is from 9.8-11.6. At this pH range no microbiology is present. The periodic, discharge from the tank into the lagoon is up to 6,200 GPD. In the winter, the lagoon is ice- covered. In the spring, algae along with process odors occur. The lagoon is covered by ice and at aeration start up in spring severe odor emission occurs. Application Task Eliminate odor and reduce BOD. Methodology Installation of the TVT-BIO 32 System in the 3 mil gal. primary treatment lagoon. Installation occurred in February. In April bio-augmentation was added following lagoon temperature increase to ~ 56oF. Impact: (following an operation period of ten months)

� Increased microbial diversity. Dominant blue-greens (cyanobacteria) has been replaced by aerobic microlife forms (bacteria, protists; microcrustacean populations). [installation + 6 months]; high MLSS.

� Total elimination in odor emissions (essentially no odor); first time in five years. � BOD loading (3,800 – 10,600 mg/L) reduced by 73%. (250 mg/L). � Sludge reduction within lagoon system by 50%.. � pH ~ 7.7. � FOG reduced from 71 to 11. � Significant biological nutrient removal (BNR25).

24 Whey or milk plasma is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacture of cheese or casein. Acid whey is obtained during the making of acid types of cheese such as cottage cheese. The whey in this case history was acid (pH = 6. 1 – 6.4). 25 Biological nutrient removal (BNR) removes total nitrogen (TN) and total phosphorus (TP) from wastewater through the use of microorganisms under different environmental conditions in the treatment process. Total effluent nitrogen comprises ammonia, nitrate, particulate organic nitrogen, and soluble organic nitrogen. The biological processes that primarily remove nitrogen are nitrification and denitrification. During nitrification ammonia is oxidized to nitrite by one group of autotrophic bacteria, most commonly Nitrosomonas. Nitrite is then oxidized to nitrate by another autotrophic bacteria group, the most common being Nitrobacter. Nitrification occurs in the presence of oxygen under aerobic conditions, and denitrification occurs in the absence of oxygen under anoxic conditions. Biological phosphorus removal relies on phosphorus uptake by aerobic heterotrophs capable of storing orthophosphate in excess of their biological growth requirements. The treatment process can be designed to promote the growth of these organisms, known as phosphate-accumulating organisms (PAOs) in mixed liquor.


Case History: Dairy Farming Background 500+ milking cows; 400 heifers; 3,000+ acre dairy farm. Manure from the barns is taken to a screw press which reduces the percent solids from 14% to 6%. (solids are then composted in a green house). Liquid manure is then pumped to a geo-covered anaerobic lagoon (#1) where methane is automatically flared. The liquid manure is gravity-fed into a series of 3 additional lagoons with aerators. Field application. Lagoon #1 (2 million gallons, geo-covered) anaerobic Lagoon #2 (135,000 gallons) aerobic Lagoon #3 (121,000 gallons) aerobic Lagoon #4 (435 gallons) aerobic (TVT BioReactor) � discharge to farmland. Application Task Eliminate odor and reduce BOD. Methodology Installation of the TVT-BIO 32 System In Lagoon #4 (clarification lagoon). Impact: (following an operation period of five months)

� DO levels increased from “0.0 mg/L” to 4.2 mg/L through the clarification lagoon. � Increased microbial diversity. Flourishing protozoan and metazoan communities. [installation + 5

months]; high MLSS. � Significant odor reduction; essentially “zero.” � Significant biological nutrient removal (BNR)


Case History: Steel Plant Background The facility WWTP (waste water treatment plant) discharges approximately 50 to 65 million GPD of process oil-contaminated wastewater into the Rouge River daily. Process wastewater is first piped into a trap tank (with a skimmer), and from there, into two sludge ponds at the WWTP where, traditionally, hand-cast microbiology is added. The first sludge treatment pond (West Sludge Pond) in where the TVT Bio-Reactor was placed. Outflow is directed over a weir before passing to the second sludge (polishing) pond, and then to the river. Each sludge pond is typically in service for 12 to 15 months before the sludge (typically 8,000 cubic yards) is removed and land filled at a yearly cost of approximately $500 M. Historically, each sludge pond is in service for 12 to 15 months before sludge removal was required at a cost of $ 400 to $500M per event. Application Task

o Reduce frequency of sludge removal. o Increase biodiversity in second (polishing) sludge pond. o Reduce flocculent26 chemical cost; increase MLSS o Reduce volume of reclaimed process oil. o Increase degradation of targeted substrates: FOG.

Methodology Installation of the TVT-BIO 32 System in the first process lagoon (West Sludge Pond). Impact: (following an operational period of eight years)

� Reduced sludge production. Sludge removal frequency is now approximately every 3 years – a decrease of 50%. Considering both ponds, this indicates an increase of 1.25 years before sludge removal. Cost savings of $750M.

� Decrease in amount of sludge removed (8,000 cu yards to ~ 4,000 cubic yards – a decrease of 50%.

� FOG (fat, oil, and grease) reduced. Previous to the introduction of process-specific microbiology, no traditional technology was able to reduce the volume of (target substrates) hydrocarbons, FOG in the sludge.

� Increased microbial diversity. Dominant blue-greens have been replaced by aerobic microlife forms (bacteria, protests; microcrustacean populations). [installation + 6 months]; high MLSS.

� The discharge back into the Rouge River is cleaner than the water taken out at the beginning for mill water to start the process for use in creating roll steel.

26 Particles finer than 0.1 µm (10-7m) in water remain continuously in motion usually due to a negative electrostatic charge that causes them to repel each other. If this electrostatic charge is neutralized by the use of coagulant chemical, the finer particles start to collide and agglomerate forming larger and heavier particles are called flocs. Flocculants are used in water treatment processes to improve the sedimentation or filterability of small particles. Many flocculants are multivalent positively-charged (cations) such as aluminum, iron, calcium or magnesium.


Case History: Food Processing (Canning) Background Although green beans are its primary offering, the canning company processes a variety of products depending on the season: asparagus (spring), green beans-kidney plus others; (May through December); apple and pumpkin (fall). In addition, the company fills in production gaps with a range of dry beans including garbanzos, kidneys, pintos, black beans, and red beans in chili sauce, among others. Installation of an unlined 8-acre wastewater lagoon (4 – 30’ in depth) occurred in 1967. The waste-stream was pumped a ¼ mile from the plant to the lagoon, which originally did not have any aeration process with simple evaporation as the discharge strategy. This proved to be an “effective solution” up until the ‘90s, when the canning operation increased its year-round dry bean production. At that point BOD levels spiked along with a significant increase in nuisance odors.

Current environmental challenges include: challenges in the form of elevated Biological Oxygen Demand (BOD) levels in the waste-stream, increased demand for dissolved oxygen (DO). Application Task

o Increase low DO levels (originally at 0.67 ppm). o Reduce high BOD levels (originally at 1,500 mg/L). o Reduce nuisance odors. o 6-8” deposition sludge layer needed to be removed at considerable cost. Achieve

remediation without removal due to concerns regarding groundwater incursion. o Increase biodiversity through bio-augmentation.

Methodology Installation of the TVT-BIO 32 System into the treatment lagoon. Impact: (following an operational period of one year)

� Do levels increased to 3.5 – 4.2 ppm27 over various portions of the lagoon. � BOD levels saw a similar change going from 1,500 mg/l to below 350 mg/l. � Essentially an elimination of nuisance odors. � MMLS increased from 104 to 109 � Introduction of site-specific microbiology to aid in lagoon sludge reduction. � Despite these improvements, the irregularly shaped lagoon — measuring about 8 acres and

varying in depth from 4’ to 30’ — still harbored some low DO areas. To eliminate these areas, TVT-bio installed a 15 HP Eductor-Venturi (EV28). In this installation at the Eductor-Venturi outlet the Dissolved Oxygen was measured at DO 11 and at a distance of 95 feet still at DO 3.5. During the last five years while the TVT- bio systems and process have been in the existing lagoon, the odor has been eliminated; the Dissolved oxygen (DO) has been increased to an average throughout the lagoon between DO 3.5 to DO 4.2 and the BOD reduced to 250 mg/l.

27 When DO achieved 4 ppm, the canning operation went so far as to introduce catfish into the lagoon. Upper DO level 6 ppm. 28 The EV is a patented scalable submersible laminar flow aeration process at 460 gallons per minute with a plume of 120 feet.


Current Research: Hypersaline29 Remediation

Oil sands are naturally occurring mixtures of sand or clay, water and an extremely dense and viscous form of petroleum called bitumen. They are found in large amounts in many countries throughout the world, but are found in extremely large quantities in Canada and Venezuela.

Oil sands reserves have only recently been considered to be part of the world's oil reserves, as higher oil prices and new technology enable them to be profitably extracted and upgraded to usable products.

Because extra-heavy oil and bitumen flow very slowly, if at all, toward producing wells under normal reservoir conditions, the sands must be extracted through the use of in situ techniques which reduce the viscosity by injecting steam into the sands. This extraction process produces an oil-contaminated brine solution that must be treated before release to the environment. TVT holds a number of proprietary hypersaline microbe strains that demonstrate a significant chloride reduction capability in benchtop test scenarios / engineering studies. These studies are refining a remediation strategy that combines both hypersaline and oil-degrading microbe strains in a bio-reactor system to reduce these two co-contaminants.

29 Water with a salinity that is greater than 40 parts per thousand (ppt). This is in contrast to freshwater which has a salinity of less than 0.5 ppt and ocean water that is typically 35 ppt.


Testimonials Comments and Excerpts Regarding TVT-bio Technology and Applications

Darrin J. Costantini P.E. Senior Engineer, Haley & Aldrich, Inc.

…“Energy savings relative to conventional “splasher” and aeration processes are tremendous, ranging from 15% to 40%…pay back periods are typically less than three years…empirical predictors have been established that indicate that the performance of the TVT-bio system is equal or greater than those of competing units on the market…“

Brian Bailey VP Operations, YANCY’S FANCY

…”Mr. Hausin’s approach intrigued me for several reasons…the possibility of reducing BOD (Biological Oxygen Demand) and odor difficulties, while…eliminating or reducing large capital intensive upgrades and associated engineering and consulting fees…we observed significant BOD reduction, no odor build-up, which has been an annual problem in spring and early summer of each year…Our wastewater is extremely variable…and there is no flow equalization mechanism…The TVT-bio System operated effectively…requiring minimum maintenance…In summary, we enjoyed working with Mr. Hausin and his team…It is my view that the TVT-bio Systems process will prove to be of significant benefit to the wastewater treatment Industry in terms of performance, cost-effectiveness & reliability.”

T.R. Barstow P.E. Area Manager, Schaeffer Road WWTP, Severstal NA

…”Historically, we have had each sludge pond in service for 12 to 15 months prior to using bacteria…with bacteria and 19 months of service…no visible indication the pond needed to be taken out of service…after 12 to 15 months the west pond would have 3,800 to 4,200 yards of sludge removed…after 19 months the survey showed 2,900 yards of sludge…delaying the cleaning…for eight additional months…this indicates an increase of 1.25 years…we would be required to clean the ponds every 3.75 years rather than every 2.5 years…a 50 % increase in time between cleaning…or a 33% reduction in frequency of cleaning ponds…reduction of cleaning ponds…reduction of reclaiming oil shipments…reduction of flocculent…could more than justify the purchase of the TVT-BioReactor…”


Selected References

Clair N. Sawyer, Perry L. McCarty, Gene F. Parkin (2003). Chemistry for Environmental Engineering and Science (5th ed.). New York: McGraw-Hill. ISBN 0-07-248066-1.

Connecticut Department of Environmental Protection (CTDEP). 2007. Nitrogen Removal Projects Financed by the CWF

through 2006. Provided by Iliana Ayala June 13, 2007. Foess, G.W., P. Steinbrecher, K. Williams, G.S. Garrett. 1998. Cost and Performance Evaluation of BNR Processes. Florida

Water Resources Journal: December 1998. Gannett Fleming. No date. Refinement of Nitrogen Removal from Municipal Wastewater Treatment Plants. Prepared for the

Maryland Department of the Environment. Online at http://www.mde.state.md.us/assets/document/BRF%20Gannett%20FlemingGMB%20presentation.pdf.

Jeyanayagam, Sam. 2005. True Confessions of the Biological Nutrient Removal Process. Florida Water Resources

Journal: January 2005. Keplinger, K.O., J.B. Houser, A.M. Tanter, L.M. Hauck, and L. Beran. 2004. Cost and Affordability of Phosphorus Removal at

Small Wastewater Treatment Plants. Small Flows Quarterly. Fall 2004, Volume 5, Number 4. Maryland Department of the Environment (MDE). 2006. BNR Costs and Status BNR Project Costs Eligible for State Funding.

Provided by Elaine Dietz on October 31, 2006. Park, Jae. No date. Biological Nutrient Removal Theories and Design. Online at

http://www.dnr.state.wi.us/org/water/wm/ww/biophos/bnr_removal.htm. Water Environment Federation (WEF) and American Society of Civil Engineers (ASCE)/Environmental and Water

Resources Institute (EWRI). 2006. Biological Nutrient Removal (BNR) Operation in Wastewater Treatment Plants. McGraw Hill: New York.


APPENDIX Company Patents United States Patent 60/909,954 Hausin , et al. US Provisional Patent Application 60/909,954 " FIXED FILM BIOPROCESS FOR REMOVING CARBON COMPOUNDS IN OIL AND GAS DRILLING SLUDGE" A system for recycling drilling sludge is also disclosed. The system has a treatment area and a salt-tolerant bio-reactor, coupled to the treatment area. The bio-reactor has at least one bio-suspension element for supporting the growth of at least one type of biological microorganism within an enclosed flow zone; an agitator for creating a flow of the drilling sludge through the enclosed flow zone at a flow rate; and an aerator for providing a gas to the enclosed flow zone. The system further has a processor coupled to the agitator and configured to create at least a minimum self-cleaning drag force between drilling sludge passing through the enclosed flow zone and the combined at least one type of biological microorganism and the at least one bio-suspension element. Related methods and bioreactors are also disclosed. United States Patent 60/941,272 Hausin , et al. US Provisional Patent Application 60/941,272 "VERTICAL LAND-BASED BIOREACTOR" A bioreactor for treating a liquid has a container comprising a top portion, a vertical wall portion, and a bottom portion, wherein said top portion comprises at least a top port that is either sealed or unsealed, and wherein said vertical wall portion comprises at least one inlet port and at least one outlet port. The bioreactor has a media support platform securely disposed inside the container at a position below the inlet and outlet ports, wherein the media support platform is substantially perpendicular to the container’s vertical wall portion and substantially spans the cross-sectional area of the container, and wherein the media support platform is

permeable to liquid. The bioreactor also has at least one cylindrical chamber securely disposed inside the container at a position above the media support platform, wherein said at least one cylindrical chamber comprises a top end, a bottom end, and a sidewall portion. The bioreactor has a plurality of media units topically disposed on the media support platform in an outer chamber portion, wherein said outer chamber portion is defined by the space between the sidewall portion of the at least one cylindrical chamber and the vertical wall portion of the container. The bioreactor further has a recirculation/aeration system secured at the bottom end of the cylindrical chamber, wherein said recirculation/aeration system comprises a submersible pump device that can be programmed to pump liquid at variable speeds and that comprises a liquid intake portion having perforations, an air intake tube, and an eductor device, and wherein the air intake tube protrudes through the top port of the container’s top portion. Related methods and conversion kits are also disclosed. United States Patent 6,022,476 Hausin February 8, 2000 Water treatment process Abstract A method for removing selected biodegradable materials from a body of water. In the first step of this method, a plurality of bio-suspension elements are disposed within an enclosure which is floating at least partially submerged in a body of water; the bio-suspension elements provide surfaces for supporting the growth of at least five different biological microorganisms. The different biological microorganisms are intermittently introduced into the enclosure along with water; the water is continuously aerated and fed into the enclosure at a rate of at least about 60 feet per minute. The treated water so produced is continuously removing from the enclosure at a rate of at least about 60 feet per minute. The pH, the total dissolved solids, the temperature, and the flow rate of the treated water is continuously measured.


United States Patent 6,231,766 Hausin May 15, 2001 Water treatment process Abstract A process for treating a body water to purify it. In this process, a portion of the water to be treated is continuously caused to flow at a rate of at least about 60 feet per minute and is continuously agitated, aerated, and fed into a biochamber within which are disposed at least five distinct strains of microorganisms. A screen is disposed in the biochamber below the microorganisms, and air is forced through such screen during the processing of the water. United States Patent 6,821,426 Hausin , et al. November 23, 2004 Process for treating a body of water Abstract A process for treating a body of water in which a bioreactor and a water eductor are located in a body of water such that the eductor is submerged beneath the body of water to a depth of at least about 3 feet. The water eductor produces a water flow at a rate of at least about 60 feet per minute; the water flow is substantially parallel to the surface of the body of water. Water is also passed through a bioreactor that contains a plurality of bio-suspension elements within an enclosure located above a screen in the enclosure.

United States Patent 7,101,483 Hausin , et al. Process for treating a body of water Abstract Aprocess for treating a body of water in which a bioreactor and a water eductor are located in a body of water such that the eductor is submerged beneath the body of water to a depth of at least about 3 feet. The water eductor produces a water flow at a rate of at least about 60 feet per minute; the water flow is substantially parallel to the surface of the body of water. Water is also passed through a bioreactor that contains a plurality of bio-suspension elements within an enclosure located above a screen in the enclosure.

TVT-Bio Bio-Reactor Technology and Applications Page | 20

GLOSSARY Activated Sludge Treatment (see also “sludges”) Activated sludge plants use a variety of mechanisms and processes that use dissolved oxygen (DO) to promote the growth of biological floc that substantially removes organic material. It also traps particulate material and can, under ideal conditions, convert ammonia to nitrite and nitrate ultimately to nitrogen gas, (see also denitrification; nitrification). Activated Carbon Highly adsorbent carbon obtained by heating granulated charcoal to exhaust contained gases, resulting in a highly porous form with a very large surface area. It is used primarily for purifying gases and liquids by adsorption.

Absorption A physical or chemical process in which atoms, molecules, or ions A physical or chemical process in which atoms, molecules, or ions enter a gas, liquid or solid material. This process differs from adsorption in that the molecules are taken up by the volume, not by surface. Adsorption The adhesion of molecules of gas or liquid, to the surface of a solid or liquid, which it is in contact with. For example, the adsorption process is used to remove a soluble substance from a liquid, by using a solid such as active carbon. Alkalinity This is a measure of a wastewater's capacity to neutralize. The bicarbonate, carbonate, and hydroxide ions are the primary contributors to alkalinity. The determination of alkalinity levels at various points in a plant will be an aid to the proper understanding and interpretation of the treatment process. For example, if chemical addition is used to coagulate wastewater for solids removal, hydrogen ions may be released and cause the pH to decrease. Alkalinity will tend to neutralize the acids formed and permit coagulation to proceed in the proper pH range. Some other processes dependent on pH are disinfection, digestion, and sludge preparation and conditioning. Anaerobic Digestion A bacterial process that is carried out in the absence of oxygen. The process can be either thermophilic digestion in which sludge is fermented in tanks at a temperature of 55°C or mesophilic, at a temperature of around 36°C. Though allowing shorter retention time, thus smaller tanks, thermophilic digestion is more expensive in terms of energy consumption for heating the sludge.

The holding time in the digester tank for mesophilic digestion is between 15-30 days. Thermophilic digestion is faster, around 2 weeks. Thermophilic is much more expensive, requires more energy, and is less stable than the mesophilic process. There are four stages to anaerobic digestion:

STAGE 1 Complex organic molecules are broken down (by a chemical process called hydrolysis) into more simple compounds (sugars, amino acids, and fatty acids). STAGE 2 Further breakdown of these simpler products into ammonia (NH3), carbon dioxide (CO2), and hydrogen sulfide (H2S) in a process called acidogenesis (making acids). STAGE 3 Further breakdown of simple products into carbon dioxide (CO2), hydrogen (H), and acetic acid (C2H4O2) in a process called acetogenesis (making acetic acid). STAGE 4 Final products of methane gas (MH4), carbon dioxide (CO2), and water H2O) are produced in a process called methanogenesis (methane gas production).

One major feature of anaerobic digestion is the production of biogas, which can be used in generators for electricity production and/or in boilers for heating purposes. Biogas is a mixture of methane (MH4), carbon dioxide (CO2), hydrogen (H), and traces of hydrogen sulfide (H2S). The solid components of anaerobic digestion vary greatly. Most are not easily disposed of. Odor generation, cost, and lack of continuous productivity are some of the mitigating factors in using the technology. Anaerobic digestion is sometimes employed before aerobic digestion for the treatment of high-strength, readily degradable wastewaters. The primary advantages of the anaerobic process are low sludge production and the generation of energy in the form of biogas. Aerobic Digestion A bacterial process occurring in the presence of oxygen. Under aerobic conditions, bacteria, and other microbes, rapidly consume organic matter and convert it into carbon dioxide. Because aerobic digestion occurs at a much faster rate than anaerobic digestion, capital costs are lower. However, operating costs are characteristically much greater for aerobic digestion because of energy costs for aeration needed to add oxygen to the process.


Examples of the aerobic digestion process include: open lagoons (e.g. paper mill pulp), fixed-film processes (e.g. trickling filters, rotating biological contactor--RBC). Aerobic Granular Reactor (sludge-generation) A type of discontinuous fed system wastewater treatment facility. Here, stable granulated sludge is created under aerobic circumstances. Simultaneously, COD, nitrogen (N) and phosphorous (P) removal can also be integrated into the system. Because of the settling capacity of the sludge granules, this type of system is very compact—about 20% of the size of a conventional activated (sludge digested with bacteria) system. Biofloc (biological floc) A microbe aggregation, especially bacteria, held together by polymeric compounds. Biofilm A complex individualized community of microbes that are attached to a substrate. Biofilms are usually found on solid substrates submerged in or exposed to some aqueous solution, although they can form as floating mats on liquid surfaces. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic. Biofilms can contain many different types of microbes, e.g. bacteria, protozoa and algae; each group performing specialized metabolic functions. However, some organisms will form monospecies films under certain conditions. Biofilms are held together and protected by polymeric compounds called extracellular polymeric substance (EPS). This chemical matrix protects the cells within it and facilitates communication among them through biochemical signals. Biological Aerated Filters (BAF) Process technology that combines filtration with biological carbon reduction, (see “nutrient removal”). BAF usually includes a reactor filled with a filter media. The dual purpose of this media is to support highly active biomass that is attached to it and to filter suspended solids. Bioaugmentation The introduction of a group of natural microbial strain(s) to achieve bioremediation. Bioremediation Any process that uses microbes, fungi, green plants or their enzymes to return the environment altered by contaminants to its original condition. BOD (Biological Oxygen Demand) A test used to measure the concentration of biodegradable organic matter present in a sample of water. It can be used to infer the general quality of the water and its degree of organic content or enrichment (e.g. pollution). BOD is not an accurate

quantitative test and should be considered as providing an indicator of the quality of a water body.

Typical BOD Values: “Pristine” Waters - < 1mg/L “Moderately” Polluted Waters – 2-8 mg/L Treated Municipal Sewage (tertiary treatment) – 20 mg/L Untreated Municipal Sewage – 2,000 – 6,000 mg/L Sewage Treatment Plant Influent – 200 mg/L

Brownian Motion Named for Robert Brown; the motion results from collisions between the grains and atoms or molecules in a fluid. Chemotaxis The movement or orientation of an organism or cell along a chemical concentration gradient either toward or away from the chemical stimulus. C:N:P Ratios Microbial cells are largely comprised of carbon (C), nitrogen (N) and phosphorus (P) at an average C:N:P ratio of 50:14:3. Sufficient amounts of these nutrients must be available in a usable form and in proper proportions for unrestricted microbial growth to occur.

Carbon Carbon is the most basic structural element of all living forms and is needed in greater quantities than other elements. Nitrogen Nitrogen is found in the proteins, enzymes, cell wall components, and nucleic acids of microorganisms. Because molecular nitrogen (nitrogen gas) can be used by only a few microbe types, most microbes require a chemically- “fixed” form of nitrogen, such as ammonia (NH3), nitrite (NO2) or nitrate (NO3). Phosphorous Phosphorous is needed for construction of cell membranes (composed of phospholipids), in ATP (energy source of cell) and to link together nucleic acids.

COD (Chemical Oxygen Demand) A chemical test commonly used to indirectly measure the amount of organic compounds in water making COD a useful measure of water quality. It is expressed in milligrams per liter (mg/L), which indicates the mass of oxygen consumed per liter of solution.

Comparing COD to BOD:

• COD Faster process control know what you are sending downstream within two hours rather than five days.

• COD is a more stable measurement method


• COD is more stable than BOD, the tests use different methods of oxidation. BOD uses microbes; COD uses a chemical (potassium dichromate).

• COD values are usually slightly higher than corresponding BOD values.

Denitrification (compare with nitrification) A critical biological process in which certain bacteria convert nitrates (NO3) and nitrites (NO2) to atmospheric nitrogen (N). Without biological denitrification, the earth's nitrogen supply would eventually accumulate in the oceans, since nitrates are highly water soluble and are continuously leached from the soil into nearby bodies of water. Denitrifying bacteria include many species of Pseudomonas, Alkaligenes and Bacillus. Diffusion Diffusion is the movement of molecules from a high concentration to a low concentration. Many molecules diffuse across cell membranes.

Dissolved Oxygen Amount of gaseous oxygen (O2) actually present in water expressed in terms either of its presence in the volume of water (milligrams of O2 per liter – mg/L) or of its share in saturated water (percentage). Eductor-Venturi A TVT-patented system for moving fluids and mixing them with air. Filtration Secondary treatment process that removes suspended particulate matter. Filtration over activated carbon removes residual chemical contaminants. Filter Beds (oxidizing beds) Trickling filter beds are used where sewage is spread onto the surface of a deep bed made up of coke (carbonized coal), limestone chips or specially fabricated plastic media. Such media must have high surface areas to support the biofilms that form. Final Treatment Treatment process that focuses on removal of disease-causing organisms from wastewater. Treated wastewater can be disinfected by adding chlorine, ozone, or by using ultraviolet light. Fixed Film Systems Grow microbes (“microbiology”) on substrates such as rocks, sand or plastic. The wastewater is spread over these substrates,

allowing the wastewater to flow past the film of microorganisms fixed to the substrate. This provides an extremely efficient way of allowing microbes an opportunity to feed on dissolved nutrients. Trickling filters, rotating biological contactors, and sand filters and aquarium filters are examples of fixed film systems. Fixed Film BioReactor (TVT-bio) Patented process aerobic systems that combine specific microbe biofilm technology to bioremdiate dairy, winery, agricultural, and industrial process waste streams. • Installation in existing lagoons or on land without cranes or

special equipment. • Operation with minimum monthly maintenance. • Controlling nitrification—conversion of ammonia to nitrates. • Increasing dissolved oxygen to reduce biological oxygen

demand. • Controlling of fats, oils, and grease (FOG) • Reduction in treatment timer. • Odor control (within 1 month of startup). • Sludge reduction (3050%). • Increased oxygen transfer increasing microbial processing

efficiency. • Lower operating costs (reductions of electricity and

chemical use). FOG Fats, oils, and grease. Heterotrophc Plate Count The lower the HPC, the better the biological water quality. Other names for the procedure (within the water industry) include total plate count, standard plate count, plate count and aerobic plate count.Although standardized methods have been formalized, HPC test methods involve a wide variety of test conditions that lead to a wide range of quantitative and qualitative results. Temperatures employed range from around 20°C to 40°C, incubation times from a few hours to seven days or a few weeks, and nutrient conditions from low to high. The test itself does not specify the organisms that are detected. Hydrophobic Repelling, tending not to combine with, or incapable of dissolving in water. Open Lagoon Systems Shallow basins (usually no more than 2 meters (6 feet) that hold the wastewater for several months to allow for the natural degradation of sewage. These systems take advantage of natural aeration and microbes present in the wastewater to process sewage. Lagoons are highly aerobic and colonization by native macrophytes, (aquatic plants) especially reeds. Small filter


feeding microinvertebrates such as Daphnia and rotifers greatly assist in treatment by removing fine particulates. Lagoon, aerated If compressed air is introduced to an open lagoon (using an aerator or diffuser – a producer of tiny bubbles) -- the rising column of air bubbles moves bottom water to the surface where it is exposed to the atmosphere. During this uplift process, large volumes of water will loose bad gasses to the atmosphere, and at the same time, pick up oxygen (at the water/oxygen interface) while on the surface. Lagoon, anaerobic Detention area or basin of wastewater that uses anaerobic microbes for digestion. A minimum of 2 meters (6 feet; not exceeding 6 meters (36 feet)) is required for an anaerobic lagoon. If the lagoon system is being used for energy production (e.g. biogas), a cover is placed over the lagoon. The cover catches biogas produced by anaerobic bacteria. Locales with cold winters are inappropriate for anaerobic lagoons because these organisms are sensitive to lower temperatures. Media Polypropylene packing media, which act as a substrate for biofilm growth.

Image © Lantec

Membrane Biological Reactors (MBR) Process utilizes a semi-permeable membrane barrier system either submerged or in conjunction with an activated sludge process. This technology guarantees removal of all suspended and some dissolved pollutants. The limitation of MBR systems is directly proportional to nutrient reduction efficiency of the activated sludge process. The cost of building and operating a MBR is usually higher than conventional wastewater treatment. Microbes (“microbiology”) A “catch-all” term that refers to bacteria, microfungi (not mushrooms), protests, and microinvertebrates (e.g. Daphnia

and rotifers) that feed on suspended organic matter and/or other carbon sources. Wastewater treatment process microbes include:

Bacteria: Image © Wim van Egmond E-mail: [email protected]

Bacteria are responsible for the stabilization of wastes coming into a treatment plant. Many of these bacteria form flock particles, or clusters of bacteria that break down waste. Protozoa:

Image © Wim van Egmond E-mail: [email protected]

Protozoa are microscopic, unicellular organisms. They are found in large numbers in the activated sludge process. Protozoa perform many beneficial roles in the treatment process, including the clarification of the secondary effluent through the removal of bacteria, the flocculation of suspended material and as bio-indicators of the health of the sludge. Microinvertebrates Rotifers: Image © Wim van Egmond E-mail: [email protected]

Rotifers


are the most abundant microinvertebrates found in the activated sludge process. Rotifers move by swimming freely or crawling. Micro invertebrates Nematodes Image © Wim van Egmond E-mail: [email protected]

Nematodes are aquatic animals present in fresh, brackish waters, salt waters, and soil worldwide. Freshwater nematodes can be present in sand filters and aerobic treatment plants.

Microbe Diversity In most natural environments there are large numbers of microbes present. In these healthy aquatic environments there can be 105 - 107 (100,000 - 10,000,000) organisms per mL. In surface soils the number of microbes can range from 107 - 109 per gram. It is important to recognize that often only a fraction of the total microbial community may actually metabolize (“eat”) the compound(s) of concern and a majority of organisms utilize other carbon sources that are present. Numerous factors can influence the distribution and numbers of microorganisms found at a particular site. Moreover, organisms able to degrade some contaminants are widely distributed in nature and are likely to be found at most sites, whereas organisms able to degrade more unique compounds may not be widespread. Prevailing site conditions such as high concentrations of contaminants or toxicants (heavy metals, for example) and other factors such as extremes of pH, low moisture, and nutrient limitation, can restrict or preclude microbial growth. In some instances, the rate of biological degradation can be increased through the addition of microbes that have been shown to degrade the contaminants of concern at high rates or are particularly well suited to remain active under prevailing site conditions. This process is referred to as bioaugmentation. For example, a lagoon filled with whey waste may have an organism count of 102 organisms. Following application of fixed biofilm technology, an organism count of 107 can be expected.

Moving (Rotating) Bed Biological Reactor (MBBR) see also Rotating (Rotary) Biological Contactor (RBC) Involves the addition of moving inert media into existing activated sludge basins to provide active sites for microbe film creation and attachment. Nitrification The biological conversion (called oxidation—adding oxygen) of toxic ammonia (NH4) using oxygen into nitrite (NO2), followed by further oxidation, to nitrates (NO3). This biological oxidation of ammonia to nitrate is performed by nitrifying bacteria. The first step in converting ammonia to nitrite is done by a host of Nitrosomonas and Nitrosococcus bacteria. The second step, oxidizing nitrite to nitrate, is done by Nitrobacter bacteria. Nitrification plays an important role in removing nitrogen from wastewater. Nutrient Removal Wastewater may contain high levels of the nutrients nitrogen and phosphorus. Excessive release to the environment can lead to a build up of these and other organic nutrients, called eutrophication, which can in turn encourage the overgrowth of weeds, algae, and cyanobacteria (blue-green algae). So much algal/plant matter can be present that the consumption of dead plant matter by bacteria (decay) creating odors and depleting oxygen levels in the water suffocating fish and other aquatic life. Different treatment processes are required to remove nitrogen and phosphorus.

- Nitrogen Removal: Most nitrogenous wastes begin as ammonia. Special bacteria convert ammonia into nitrite. Still other bacteria convert nitrite into nitrate. Other bacteria convert nitrates into nitrogen gas (a process called nitrification). Sometimes the conversion of toxic ammonia to nitrate alone is referred to as tertiary treatment.

- Phosphorus removal: (Biological) - Phosphorus can be removed biologically in a process called enhanced biological phosphorus removal. In this process specific bacteria, called PAC’s (Polyphosphate Accumulating Organisms). PAC’s accumulate phosphorous in their cells. When the biomass enriched in these bacteria is separated from the treated water, these biosolids have a high fertilizer value. (Chemical) – Iron compounds (ferric chloride) or alum (aluminum phosphate) can chemically precipitate phosphorous.


pH Substance pH Acid mine runoff -3.6 – 1.0 Battery acid -0.5 Gastric acid 1.5 – 2.0 Lemon juice 2.4 Cola 2.5 Vinegar 2.9 Orange or apple juice 3.5 Beer 4.5 Acid Rain <5.0 Coffee 5.0 Tea 5.5 Milk 6.5 Pure water 7.0 Healthy human saliva 6.5 – 7.4 Blood 7.34 – 7.45 Sea water 8.0 Hand soap 9.0 – 10.0 Household ammonia 11.5 Bleach 12.5 Household lye 13.5

Phosphorous (P) / Phosphate (PO4) Removal An anaerobic process whereby special bacteria (polyphosphate-accumulating organisms (PAO)) accumulate phosphorous as polyphosphate (e.g. Sodium tri-polyphosphate (Na5P3O10)). This phosphorous/phosphate fraction is generally about 6% of their biomass. Besides processing phosphate, many PAO’s can consume simple carbon compounds without the presence of nitrogen or oxygen—the reason for using them in anaerobic treatment systems. Examples of PAO’s are: Betaproteobacteria, Accumulibacter, and Actinobacteria. In most treatment plants, a chemical removal of phosphorous is usually done using aluminum sulphate (alum). It reacts to form non-soluble compounds that then settle out in the sludge. Preliminary Treatment A pre-treatment process that screens out, grinds up, or separates debris (sticks, rags, large particulate matter etc.) prior to further processing. Primary Treatment A physical treatment process that separates suspended solids and greases from wastewater. Wastewater is held in a quiet tank for several hours allowing the particles to settle to the bottom and the greases to float to the top. The solids drawn off the bottom and skimmed off the top receive further treatment as sludge. The clarified wastewater then flows on to the next stage of wastewater treatment called secondary treatment.

Rotating (Rotary) Biological Contactor (RBC) Used in wastewater treatment as a secondary process. It involves allowing wastewater to come into contact with a biological medium (media and biofilm) to remove contaminants. An RBC consists of a series of plastic discs (media) mounted on a drive shaft which is contained in a tank. The shaft is aligned with wastewater flow so that the discs rotate at right angles to the flow. About 40% of the surface area of each disc is submerged. Rotation provides aeration. Secondary Treatment A biological process that removes dissolved organic matter from wastewater. Microorganisms (microbes) are cultivated and added to the wastewater. The microbes (“microbiology”) use the organic matter present as their main carbon source (food source). Three approaches are used to accomplish secondary treatment; fixed film, suspended film and lagoon systems. Sequence Batch Reactor (SBR) Industrial processing tanks that treat outputs from anaerobic digesters in batches. Typically, oxygen is bubbled through the wastewater batch to reduce BOD and COD. There are four stages to treatment: filling, aeration, settling, and decanting. Sewage The wastewater released by residences, businesses and industries in a community. It is 99.94 percent water, with only 0.06 percent of the wastewater dissolved and a measure of the acidity of a solution, in terms of activity of hydrogen ions (H+). The pH scale is a reverse logarithmic representation of relative hydrogen proton (H+) concentration. On the pH scale, a shift up in value by one number


represents a ten-fold decrease in value. For example, a shift in pH from 2 to 3 represents a decrease in total concentration of ten times

less H+ concentration, and a shift from 2 to 4 represents a one-hundred fold decrease (10 X 10) in H+ concentration.

suspended solid material. The cloudiness of sewage is caused by suspended particles (100 to 350 mg/l).

A measure of the strength of the wastewater is biochemical oxygen demand, or BOD. Untreated sewage has a BOD ranging from 100 mg/l to 300 mg/l.

Sewage treatment is a multi-stage process to renovate wastewater before it reenters a body of water, is applied to the land or is reused. The goal is to reduce or remove organic matter, solids, nutrients, disease-causing organisms and other pollutants from wastewater. Slow Sand Filter (SSF) Used in water purification and wastewater treatment processes. The sand bed is usually 1-2 meters in depth with a flow rate of approximately 0.1 to 0.2 meters per hour (cubic meters per square meter per hour). SSF’s utilize biofilms as a biological method of processing wastewater. Many municipal water treatment facilities will have 12 or more sand beds to process wastewater. SSF’s loose performance as the biofilm grows and reduces flowthru. Rapid Sand Filter (RSF) Commonly used in municipal water treatment facilities. Here water flows under gravity or pressure through a sand filter bed. RSF’s use sand as a mechanical, not biological, filtering medium. They must be cleaned often to maintain a high flow rate. An RSF is not provide for adequate wastewater treatment on its on. Sludges Sludges are particulate-laden materials generated through the sewage treatment process. Untreated sludges are about 97 percent water. Water can be removed from sludge by using sand drying beds, vacuum filters, filter presses, and centrifuges resulting in sludges with between 80-50 percent water. This dried sludge is called a sludge cake. Primary sludges- larger particulate material that settles out during primary treatment, often have a strong odor and require treatment prior to disposal. Secondary sludges- finer particulate material containing mostly microbes from a biological treatment processes. Solids Determinations Laboratory determinations of suspended solids (SS) in the influent, primary effluent, and final effluent are standard measurements used to indicate treatment plant efficiency. The SS measurements are used in calculating the sludge volume index (SVI) and sludge density index (SDI) - both important control tools. There is a distinction between total suspended solids (TSS) and total volatile suspended solids (TVSS). TSS measures both the active bacterial mass and the inert materials in the waste or mixed liquor. TVSS is a more accurate estimate of the mass of active microorganisms in the mixed liquor and is the parameter to be used in calculating the food-to-microorganism (F:M) ratio.

Sludge Density index To determine what the return sludge pumping rate should be and to get some idea of sludge settling characteristics, sludge indices have been proposed. One of the most common is the Donaldson Index, SDI:

SDI = MLSS( %) x 100___________ % volume MLSS after 30-min settling These indices relate the weight of sludge to the volume the sludge occupies. They show how well the liquids-solids separation part of the activated sludge system is performing its function on the biological floc that has been produced and is to be settled out and returned to the aeration tanks or wasted. The better the liquid-solids separation is, the smaller will be the volume occupied by the settled sludge and the lower the pumping rate required to keep the solids in circulation. Suspended Film Systems These process systems stir and suspend microbes in wastewater. As the microbes feed on suspended organic matter and nutrients from the wastewater they grow in size and number. After the microbes have been suspended in the wastewater for several hours, they are settled out as sludge. Some of the sludge is pumped back into the incoming wastewater to provide "seed" microorganisms. The remainder is sent on to a sludge treatment process. Activated sludge, extended aeration, oxidation ditch, and sequential batch reactor systems are all examples of suspended film systems. Tertiary Treatment (effluent polishing) Used in some treatment systems to remove nutrients from wastewater. Chemicals are sometimes added during the treatment process to help settle out or strip out phosphorus or nitrogen. Some examples of nutrient removal systems include coagulant addition for phosphorus removal and air stripping for ammonia removal. Disinfection is always the final part of this process (see “final treatment). TOC (Total Organic Carbon) The amount of carbon bound in an organic compound and is often used as a non-specific indicator of water quality. The organic carbon determination is free of many of the variables involved in the COD and BOD analyses, with somewhat more reliable and reproducible data being the result. The need for rapid determination of wastewater strength has led to the development of organic carbon analyzers and their introduction into some treatment plant laboratories. All of the available instruments measure the organic carbon content of aqueous samples, although there are several methods by which this is accomplished. The TOC values will generally be less than COD values, because a number of organic compounds may not be oxidized in the total organic carbon analysis. Typical values of TOC for domestic waste range from 100 - 300 mg/L.


TOC versus BOD & COD: - TOC test does not differentiate between compounds with the same number of carbon atoms and will thus produce different oxygen demand results. TOC is NOT a replacement for COD or BOD tests.

Total Oxygen Demand (TOD) Another method of measuring organic matter in wastewater involves the oxidation of the sample to stable end products in a platinum-catalyzed combustion chamber. Total oxygen demand is determined by measuring the oxygen content of the inert carrier gas, nitrogen. TOD measurements are becoming more popular because of their quickness in determining what is entering the plant and how the plant is responding. Analysis time is approximately 5 minutes. The results obtained generally will be equivalent to those obtained in the COD test. Trickling Filter (TF) A trickle filter is one of the more efficient wastewater filtration systems. With this system, wastewater is spread (usually by a rotating perforated arm) onto the surface of a deep bed of coke (carbonized coal), limestone chips, or specially designed plastic media. The distributed wastewater trickles through this bed. The purpose of this gravity-fed media system is to encourage biofilm growth by having a constant source of food molecules (carbon and/or nitrogen) along with an abundant supply of oxygen.

An aquarium filter is a good example of a trickling filter. Water is discharged at the top, usually over a filter material that traps larger particles. Water then flows by gravity, downward, over a 6-12-inch layer of loosely-packed gravel. Aeration is provided by the water mixing with air. The gravel provides for an increased surface area for biofilm growth. The water is collected at the bottom in a sump and pumped back out into the aquarium. TSS (Total Suspended Solids) A water quality measurement that refers to the dry-weight of particles trapped by a filter, typically of a specified pore size. Values are expressed as mg/L. Wastewater Quality Indicators Biochemical oxygen demand (BOD) and the chemical oxygen demand (COD) are essentially laboratory tests to determine whether or not a specific wastewater will have a significant adverse effect upon fish or upon aquatic plant life. Whey A by-product of the cheese making process and a major component of dairy wastewater. Traditionally, it was returned back to farmer as animal feed or to spread in the fields. A large cheese factory producing 60 tons of cheese/day discharges about 150 gallons of whey/day. Whey has a high biological oxygen demand (BOD). Waste stream pH from Whey operations generally ranges from 4.6 to 6.0 (acid).


TVT Bio-Reactor

B Bio-Reactor instrumentation

Bio-Reactor field installation Installed Bio-Reactor at cheese/dairy product manufacturing lagoon

Winter conditions at cheese manufacturer’s lagoon

TVT’s Bio-Reactor is rugged and portable

Documents

Bio-Reactor Technology and Applications – a White Paper · TVT Bio-Reactor Technology and Applications | 6 Biofilms – the engine of bioremediation Biofilms are microbial populations