# Patent

## Title
Bacterial cultivation system for growth of substrate specific micro-organisms for use in industrial wastewater remediation

## Abstract

A waste treatment method includes the concentration of selected strains of bacteria in a selected medium in the presence of nutrients and water, under aerobic conditions. This concentrated batch is discharged for downstream applications in wastewater remediation. A cultivation chamber having inlet ports and a circular vent port allows for adequate air introduction and heat release. Aeration is achieved by recirculation of the fluid medium from the top of the apparatus through a pipe that runs the length of the inner wall and is specially configured at the top to minimize cell damage. Fluid can be routed tangentially in clockwise and counterclockwise directions. The conical bottom has an orifice allowing for recirculation of the fluid medium tangentially to the sidewalls. Upon completion of the batch cultivation, the medium and bacteria are discharged for downstream applications in wastewater remediation of paper mill, chemical plant, oil refinery, and other industrial effluents.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation of U.S. patent application Ser. No. 12/261,883, filed Oct. 30, 2008, now U.S. Pat. No. 8,052,873, which is a nonprovisional application of U.S. Provisional Patent Application Ser. No. 60/984,228, Oct. 31, 2007, each of which is incorporated herein by reference.
Priority of U.S. Provisional Patent Application Ser. No. 60/984,228, filed Oct. 31, 2007, incorporated herein by reference, is hereby claimed.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable
REFERENCE TO A “MICROFICHE APPENDIX”
Not applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to bioremediation and to an improved method and apparatus for bacterial cultivation, preferably for growth of substrate specific micro-organisms that are then used in industrial wastewater remediation. More particularly, the present invention relates to an improved method and apparatus for cultivating strains of bacteria in various medium (e.g. nutrients and water), under aerobic conditions and thereafter discharging the combination of concentrated bacteria and medium downstream to a reservoir that contains wastewater to be treated. The aeration and circulation of fluids is designed to limit cells shearing and damage yet achieve critical cell mass (between about 1×107 and 1×1012 cfu (colony forming units) per milliliter and dissolved oxygen levels during cultivation. Critical cell mass is the minimum number of bacterial cells per milliliter required to achieve effective bioremediation.
2. General Background of the Invention
The remediation of industrial wastewater has in the past employed various bacteria. One application where this desired remediation is particularly useful is in the pulp and paper industry. The pulp and paper industry in the United States is one of the largest fully integrated industries in the world. Each year, mills in every part of the country produce millions of tons of paper and paper products for domestic and foreign use. The Environmental Protection Agency estimates the total value of shipments from the pulp and paper industry as close to $135 billion, as much as the petroleum refining industry.
Pulp and paper manufacturing involves a series of steps, each producing one or more characteristic wastes. A typical pulp and paper mill discharges from 25,000 to over 100,000 liters of wastewater for each air-dried ton of pulp produced. While the wastewater is discharged into the environment only after it has received on-site treatment, it still contains contaminant substances and residual organic solids. Mills discharging liquid waste into rivers and coastal waters are required, pursuant to the Waste Management Act, to obtain site-specific effluent discharge permits. Because of the potential for fines and the possibility of temporary or permanent closure, maintenance of the wastewater treatment is of great importance to owners and operators within the industry. The following tables summarize the typical processes and associated contaminants with paper manufacturing.







TABLE 1







Typical Paper Industry Operations:


Materials Used and Hazardous Wastes that Might be Generated









Process/

General Types of


Operation
Materials Used
Waste Generated





Chemical
Acids/alkalies, lime,
Acid/alkaline waste


Pulping
sulfurous acid, sodium




hydroxide, sodium




sulfide



Bleaching
Chlorine bleaches,
Toxic wastewater



sulfate bleaches,
and wastewater



chloroform, solvents
treatment sludge,




Acid/alkaline waste


Papermaking
Pigments
Wastewater




treatment sludge


Sizing and
Waxes, glues, synthetic
Toxic waste,


Starching
resins, hydrocarbons
including




wastewaters and




sludges


Coating,
Inks, paints, solvents
Solvent waste, ink


Coloring, and
rubbers, dyes
waste, paint waste,


Dyeing

ignitable waste,




toxic waste


Cleaning and
Tetrachloroethylene,
Solvent waste,


Degreasing
Trichloroethylene,
toxic rinse water



methylene chloride,




trichloroethane, carbon




tetrachloride
















TABLE 2





Paper Industry Waste Descriptions
















Waste Type
Designations/Trade Names





Spent Solvents
Other Toxic or Ignitable Wastes


Carbon
Carbon Tetrachloride, Carbon Tet,


Tetrachloride
Tetrachloromethane


Methylene Chloride
Methylene Chloride, Dichloromethane


Tetrachloroethylene
Tetrachloroethylene, Perchloroethylene,



PCE


1,1,1-
1,1,1-Trichloroethane, 1,1,1-TCA


Trichloroethane



Trichloroethylene
Trichloroethylene, TCE


Chloroform
Chloroform


Benzene



Ethylene Dichlonde
Ethylene Dichloride, 1,2-Dichloroethane


Chlorobenzene
Chlorobenzene, Monmhlombenzene, Phenyl



Chloride


Methyl Ethyl
Methyl Ethyl Ketone, Methyl Acetone,


Ketone-
Meetco, But@one, Ethyl Methyl Ketone,



MEK, 2-Benzene


Mixed Spent
Halogenated Solvents


Petroleum
Petroleum Distillates


Distillates












Waste Type
Hazard Clan
UN/NAID Number





Waste Carbon
ORM-A
UN1846


Tetrachloride




Waste
ORM-A
UN1593


Dichloromethane




Waste
ORM-A
UN1897


Tetrachloroethylene




Waste 1,1,1-
ORM-A
UN2831


Trichloroethane




Waste
ORM-A
UN1710


Trichloroethylene




Waste Chloroform
ORM-A
UN1888


Waste Benzene
Flammable
UN1114


(Benzol)
Liquid 2



Waste Ethylene
Flammable
UN1184


Dichloride
Liquid



Waste Chlorobenzene
Flammable
UN1134



Liquid



Waste Methyl Ethyl
Flammable
UN1193


Ketone
Liquid



Hazardous Waste:
ORM-E
NA9199


Liquid, NOS




Waste Petroleum
Flammable
UN1268


Distillate
Liquid




Combustible
UN1268



Liquid 4











Designations
Trade Names





Corrosive Wastes



Ammonium Hydroxide
Ammonium Hydroxide, Aqueous Ammonia,



Ammonia Water, Spirit of Hartshom


Hydrobromic Acid
Hydrobromic Acid


Hydrochloric Acid
Hydrochlonc Acid, Muriatic Acid


Hydrofluoric Acid
Hydrofluoric Acid


Nitric Acid
Nitric Acid, Aquafortis


Phosphoric Acid
Phosphoric Acid, Orthophosphoric Acid


Potassium Hydroxide
Potassium Hydroxide, Caustic Potash


Sodium Hydroxide
Sodium Hydroxide


Sulfuric Acid
Sulfuric Acid, Oil of Vitriol


Other Wastes and
General Classifications


Paint Waste with
Corrosive Liquid; Corrosive Solid;


Heavy metals
Ignitable Wastes, NOS; Hazardous Wastes,



NOS


Paint Waste with
Corrosive Liquids; Corrosive Solids;


Heavy Metals
Ignitable Wastes, NOS









Within the industry, bacteria and their enzymes, along with some fungi and critical nutrient additives, have proven to be effective agents for in-situ remediation of organic wastes and subsurface pollution in soils, sediments and wastewaters associated with these processes. Effective management of a microbiological population can provide both short-term or long-term effluent improvements meeting tightening environmental restrictions, while minimizing capital expenses.
Environmental professionals are expected to “do more with less” by squeezing every ounce of performance out of the wastewater treatment system. In some cases, the quality or quantity of influent to the system has changed so much that the treatment system's design is no longer adequate to achieve the desired results. In other cases, the treatment system's capabilities have deteriorated while effluent requirements have become more stringent. In either case, innovative approaches may be necessary to allow the mill to simultaneously meet its environmental requirements, while also realizing its financial goals. Traditional waste treatment control strategies have focused on monitoring and controlling system parameters. Bioaugmentation involves applying biological additives to enhance the performance of secondary or biological wastewater treatment systems, focusing on managing the bacterial population (i.e. the work force) of the system.
In order to optimize the performance of the microbiological population, a comprehensive approach must be used to manage the system. Understanding how mill upsets and operational problems affect the microbiological population is critical to optimizing the wastewater treatment plant. Bioaugmentation has been practiced since the early 1960s. Given a history that includes misapplication of additives and poor documentation of results, the technology has come to be regarded as less than scientific in some circles. In many cases, rather than actively managing the treatment system through bioaugmentation, mills have adopted the widespread belief that over time, the proper microbes will populate the system and become acclimated to its influent. This belief assumes that the indigenous population, which is introduced via routes such as windblown solids, rainwater, and the plant influent stream, will always contain the organisms that are best suited for the service. In reality, even though the natural population may develop into an acceptable one, there may be performance limitations that only can be overcome by the introduction of additional microorganisms.
BRIEF SUMMARY OF THE INVENTION
A waste treatment method includes the concentration of selected strains of bacteria in a selected medium in the presence of nutrients and water under aerobic conditions. This concentrated batch is thereafter discharged for downstream applications in wastewater remediation. The present invention employs a cultivation chamber having inlet ports and a circular vent port that allows for adequate air introduction and heat release.
Aeration is achieved by recirculation of the fluid medium from the top of the apparatus through a pipe that runs the length of the inner wall and is specially configured at the top to minimize cell damage. Fluid can be routed tangentially in both the clockwise and counterclockwise directions within the chamber. The conical bottom also has an orifice allowing for recirculation of the fluid medium tangentially to the sidewalls. Upon completion of batch cultivation, the medium and bacteria are discharged for downstream applications in wastewater remediation of paper mill, chemical plant, oil refinery, and other industrial effluents. The aeration and circulation of the fluids is designed to limit cell shearing and damage yet achieve critical cell mass (above 107, e.g. between about 107 and 1011 per milliliter(ml)), more particularly between 108 and 1010 per milliliter(ml). Omotoa; dissolved oxygen levels are probably e.g. between about 0.5 and 100 mg/L prior to cultivation. The amount of dissolved oxygen drops as the cells respire and divide. In the preferred embodiment the dissolved oxygen uptake rate (DOUR) is preferably above 10 mg/liter/hr, e.g. between about 50 and 1500 mg/liter/hr. Typically the DOUR will be between 10 mg/liter/hr and 450 mg/liter/hr during logarithmic growth.
The present invention includes a method of wastewater remediation of a volume of wastewater stored in a reservoir, comprising the steps of providing a vessel having an interior holding a volume of nutrient liquid medium, adding a volume of bacteria to the nutrient liquid medium, and repeating this step within 12-72 hours, providing a fluid transfer system that is in fluid communication with the vessel, the transfer system including an influent flow line, a pump and an effluent flow line, and repeating this step within 12-72 hours, for a first time interval, recirculating the combination of nutrient liquid medium and bacteria through a flow path that begins within the vessel interior and flows from the vessel via the effluent flow line to the pump, returning to the vessel interior via the influent flow line, during which first time interval the bacteria concentration increases to a concentration of between about 107 to 1010 cfu (colony forming units) per milliliter (ml), after the first time interval, transmitting a volume of the combination of nutrient liquid medium and bacteria to the reservoir, and repeating this step within 12-72 hours.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein:
FIG. 1 is an elevation view of the preferred embodiment of the apparatus of the present invention;
FIG. 2 is another elevation view of the preferred embodiment of the apparatus of the present invention;
FIG. 3 is a top view of the preferred embodiment of the apparatus of the present invention, taken along lines 3-3 of FIG. 2;
FIG. 4 is a top fragmentary view of the preferred embodiment of the apparatus of the present invention;
FIG. 5 is a schematic diagram of the preferred embodiment of the apparatus of the present invention showing the addition of water to the growth chamber;
FIG. 6 is a schematic diagram of the preferred embodiment of the apparatus of the present invention showing the addition of a bacteria/medium to the growth chamber;
FIG. 7 is a schematic diagram of the preferred embodiment of the apparatus of the present invention illustrating the recirculation steps;
FIG. 8 is a schematic diagram of the preferred embodiment of the apparatus of the present invention illustrating a transmission of the recirculated bacteria, medium and water mixture to a reservoir of wastewater that is to be treated;
FIG. 9 is a fragmentary view of the preferred embodiment of the apparatus of the present invention taken along lines 9-9 of FIG. 2;
FIG. 10 is a graph indicating microbial counts over time in hours for the chamber part of the preferred embodiment of the apparatus of the present invention, wherein the chamber is filled with a nutrient blend that includes molasses in one example and a nutrient blend that includes methanol in another example;
FIG. 11 is a graph indicating microbial counts over time in hours for the chamber part of the preferred embodiment of the apparatus of the present invention, wherein the chamber is filled with a blend of nutrients and bacteria and wherein the results were obtained using a commercially available autoanalyzer;
FIG. 12 is a graph indicating a test that was conducted to evaluate a commercially available autoanalyzer, comparing the data of FIG. 11 with the data of FIG. 12 obtained using both a plate count agar media and a tryptic soy agar media;
FIG. 13 is a graph indicating temperature and degree Celsius over time in hours for different mixtures of bacteria and nutrients;
FIG. 14 is a graph indicating ph over time in hours for the chamber for different microorganism and nutrient mixtures;
FIG. 15 is a graph indicating dissolved oxygen uptake over time in hours for the chamber part of the preferred embodiment of the apparatus of the present invention wherein the chamber is filled with a nutrient and microorganisms;
FIG. 16 is a graph indicating dissolved oxygen uptake rate over time in hours for different microorganism and nutrient mixtures;
FIG. 17 is a graph indicating a percentage of BOD5 removal over time in months for a paper mill aerated stabilization basin before and after implementation of the method and apparatus of the present invention;
FIG. 18 is a graph indicating BOD5 inlet and BOD5 outlet for an aerated stabilization basin over time in months;
FIG. 19 is a graph illustrating BOD5 over time in months for an aerated stabilization basin;
FIG. 20 is a graph indicating Effluent BOD5 in pounds over time in months for a recycled paper mill and illustrating a reduction BOD5 using the method and apparatus of the present invention;
FIG. 21 is a graph indicating bacteria counts over time in months for remediation of a paper mill with an aerated stabilization basin;
FIG. 22 is a graph indicating COD and BOD5 removal over time in months for remediation of a paper mill with an aerated stabilization basin;
FIG. 23 is a graph indicating soluble COD over time in months for the method and apparatus of the present invention applied to a paper product waste stream;
FIG. 24 is a graph indicating microbial count over time for the paper product waste system of FIG. 23; and
FIG. 25 is a graph indicating percent soluble COD removed over time for paper product waste stream of FIG. 23.



DETAILED DESCRIPTION OF THE INVENTION
The term “bioremediation” as used herein refers to any process of cleaning, removing, reducing, or decreasing an amount of a waste material, contaminant, pollutant, or environmentally unsafe component from matter by enhancement of a natural population of microorganisms or by adding developed microbial cultures.
The term B.O.D. as used herein refers to biochemical oxygen demand, which is a measurement of how much oxygen is needed from the environment.
FIGS. 1 and 2 show waste treatment apparatus 10 which includes a growth chamber 11 having an upper end portion 12 and a lower end portion 13. Upper end portion 12 of growth chamber 11 provides a top 14 having an opening 15 that can be fitted with cover 27. Growth chamber 11 can include a cylindrically shaped section 16 and a conically shaped section 17. The growth chamber 11 thus provides a sidewall 18 that has an inside surface 19 surrounding an interior 20 that contains water, bacteria and medium for feeding the bacteria.
Outlet port 21 communicates with discharge piping 22, which piping 22 communicates with pump 23. A filter 24 can be placed in discharge piping 22 in between outlet port 21 and pump 23. Base 25 can provide legs 26 that support growth chamber 11. Pump 23 can be attached (for example, bolted) to base 25. Flow line 31 is an influent flow line that enables water from water supply 29 to be added to the interior 20 of growth chamber 11. Flow line 31 can be provided with valve 30 for enabling a control of influent water. Flow line 31 attaches to growth chamber 11 at inlet opening 33. Inlet opening 33 can be provided with a discharge fitting 32 such as a perforated pipe that provides some aeration of water that is transmitted via flow line 31 to interior 20 of growth chamber 11. Water is added via flow line 31 to chamber 11 until the water reaches a selected fluid or water level 28.
Pump discharge flow line 34 communicates between pump 23 and tee fitting 36. Flow rate in line 34 is typically between about 55 and 90 gallons per minute. The flow line 34 and tee 36 are connected to riser 37 and influent fitting 38. In FIGS. 1, 2 and 4, the influent fitting 38 connects via pipe section 39 to tee fitting 40. Arrows 41, 42, and 43 in FIG. 4 illustrate that recirculating fluid (a combination of water, medium and bacteria) exit tee fitting 40 in two directions as illustrated by arrows 42, 43. This action prevents any substantial vortex formation which might shear or damage the bacteria. Tee fitting 40 provides aeration and circulation of the fluid which includes bacteria, medium and water while limiting cell shearing and damage, yet at the same time achieving a critical cell mass and high dissolved oxygen level during cultivation.
The cultivation is performed by recirculating fluid using pump 23 via flow lines 34, 36 with return to pump 23 via outlet port 21 and discharge piping 22. This recirculation is preferably conducted for several hours (typically more than 4-6 hours, preferably between about 6 hours and 72 hours, more specifically between about 8 and 24 hours.
During this time, the recirculation and aeration in combination with a growth medium produces exponential growth of bacteria within the interior 20 of growth chamber 11 (see Microbial Count in FIG. 10). After a selected time period, valve 47 is opened so that the combination of water, growth medium and bacteria can be discharged via flow line 46 to discharge 48 and into reservoir 45 in the direction of arrow 49 as shown in FIG. 8. This discharge can be via gravity flow through flow lines 22, 46. Alternatively, the mixture 50 of bacteria, water and medium can be transmitted to flow line 46 using pump 23, wherein valves 35, 47 are opened.
EXAMPLES
Example 1
In the first example shown in FIG. 10 (Molasses) the chamber 11 was filled with 85 gallons of water, 500 ml of pure molasses, 80 ml of a nutrient blend of nitrogen and phosphorus, commercially available from Environmental Business Specialists, Inc (www.ebsbiowizard.com), and 320 ml of LiquiStar EE (microbe blend including various strains of bacillius and commercially available from Environmental Business Specialists, Inc (www.ebsbiowizard.com)). In the second example shown in FIG. 10 (Methanol) the chamber 11 was filled with 85 gallons of water, 250 ml MEOH, 80 ml of a blend of nitrogen and phosphorus—commercially available from Environmental Business Specialists, Inc (www.ebsbiowizard.com) and 250 ml of MicroStar L1 (microbe blend including various strains of bacillus). In both cases critical cell mass was achieved before 24 hours. For the molasses experiment the maximum microbial count achieved using the IME kool kount autoanalyzer was 1.85×109 at the 10 hour time point. For the methanol experiment, the maximum microbial count achieved at the 16 hour time point was 1×109. In both cases the logarithmic microbial growth achieved is more than adequate for downstream remediation of industrial wastes.
Example 2
In the example shown in FIG. 11 the chamber 11 was filled with 5 lbs MicroStar (a blend of various strains of bacteria including bacillius and pseudomonas together in a dry blend (e.g. brewer's or distiller's grain as a carrier) commercially available from Environmental Business Specialists, Inc (www.ebsbiowizard.com)) and 85 gallons of water. These data are an average of various experiments conducted using the MicroStar microbial augmentation product. These counts were achieved using the commercially available IME kool kount autoanalyzer (www.imeinc.com). The test was ran at two hour increments beginning at time 2 hrs and ending at time 24 hrs. On average the 2 hr counts were approximately 2e7 at experiment initiation, with logarithmic growth beginning between about 4 and 5 hours and lasting to between about 24 and 26 hours. The average microbial count achieved using this product was between about 9e9 and 1e10 The average time to achieve this cell mass is between about 12 and 36 hours, more specifically 8 and 24 hours.
Example 3
In the example shown in FIG. 12 a test was conducted to evaluate the IME kool kount analyzer against the heterotrophic plate count method including both PCA (plate count agar) media and TSA (tryptic soy agar) media. These tests were conducted over a 24 hour period with sampling every 2 hours beginning at 2 hr time point. For the evaluation using PCA, the lag phase lasted approximately 6 hours with logarithmic growth lasting approximately 14 hours starting between about 6 and 7 hours and ending between about 18 and 20 hours. The maximum microbial count utilizing the PCA media was 1e10. For the evaluation using TSA media, the lag phase lasted approximately 6 hours followed by logarithmic growth lasting approximately 15 hours starting between about 6 and 7 hours and ending between about 18 and 22 hours. The maximum microbial count utilizing the PCA media was 2.2e10.
These various evaluations of the biogenerator have proven that this system of growing up various strains of bacteria in a liquid or dry formulation give concentrations of microbes that, when released for downstream application, are in densities that enable bioremediation of various industrial wastes.
This system also allows for the effective heat release thus allowing the temperature to stay well within the preferred ranges of the microorganisms implemented (e.g. between about ambient and 40 degrees C.) (see FIG. 13). The pH is also well maintained throughout the exponential growth phase allowing for optimal growth conditions (e.g. pH between about 4 and 9, more particularly between about 6.5 and 8.5) (see FIG. 14). These evaluations also show that the aeration in this design is adequate, as indicated by the Dissolved Oxygen uptake rate (see FIG. 15). The Dissolved Oxygen Uptake Rate during logarithmic growth (between about 50 and 200) is most impressive when compared to the prior art (see FIG. 16). Dissolved oxygen uptake rates are indicative of microbial growth. As the population of microbes increases, so does the Dissolved Oxygen Uptake Rate.
In the example of FIGS. 17-19, the test data is for an Aerated Stabilization Basin (ASB) that became nonfunctional in 2006-2007 due to excessive solids buildup and inversion of solids. The method and apparatus of the present invention were implemented to maximize and optimize performance of the Aerated Stabilization Basin under current limitations including limited aeration and loss of retention time due to solid buildup. To accelerate biological reduction of solids inventory in the Aerated Stabilization Basis, five (5) pounds per day of bacteria was applied via the apparatus 10 of the present invention as shown and described in FIGS. 1-9. These data depict an increase in the percent of BOD5 removal after implementation of the method and apparatus of the present invention beginning in August, 2007. All solid straight lines depict data gaps.
In FIG. 18, the same Aerated Stabilization Basin as treated by apparatus 10 of the present invention shows BOD5 inlet and BOD5 outlet values. These values demonstrate that there is a significant decrease in the outlet BOD5 when compared to the inlet BOD5.
FIG. 19 shows final results as respect to final effluent BOD5 for the same Aerated Stabilization Basin that was treated as described with respect to FIGS. 17 and 18. FIG. 19 illustrates stabilization of BOD5 for basin 4. FIG. 21 depicts the microbial counts (cfu/ml) achieved in two bacterial acceleration chambers 11.
In FIGS. 20-22, test results are directed to the waste treatment and remediation at a recycle paper mill that discharges to the local POTW treatment. This local POTW treatment has been unable to meet BOD5 permanent restrictions. Bioaugmentation was implemented using the method and apparatus of the present invention resulting in compliance with BOD5 permanent restrictions for nine consecutive months. FIG. 22 illustrates removal of both BOD5 and COD from the wastewater basin.
FIG. 23 is a case study for a paper products/chemical company that provides products to the paper industry. The method and apparatus of the present invention were added to the treatment system in place on or about October, 2007 date. The apparatus 10 of the present invention was effective in growing bacteria to maximum yield, enabling a reduction in COD and BOD5.
FIG. 24 illustrates the microbial count (cfu/ml) achieved. FIG. 25 illustrates the percent COD removal from the wastewater basin.
The following is a list of parts and materials suitable for use in the present invention.










PARTS LIST








Part Number
Description





10
waste treatment apparatus


11
growth chamber


12
upper end portion


13
lower end portion


14
top


15
opening


16
cylindrical section


17
conical section


18
side wall


19
inside surface


20
interior


21
outlet port


22
discharge piping


23
pump


24
filter


25
base


26
leg


27
cover


28
fluid level/water level


29
water supply


30
valve


31
flow line


32
discharge fitting


33
inlet opening


34
pump discharge flow line


35
check valve


36
tee fitting


37
riser


38
influent fitting


39
pipe section


40
tee fitting


41
arrow


42
arrow


43
arrow


45
wastewater reservoir


46
flow line


47
valve


48
discharge


49
arrow


50
mixture









All measurements disclosed herein are at standard temperature and pressure, at sea level on Earth, unless indicated otherwise. All materials used or intended to be used in a human being are biocompatible, unless indicated otherwise.
The foregoing embodiments are presented by way of example only; the scope of the present invention is to be limited only by the following claims.

## Claims

1. A method of wastewater remediation of a volume of wastewater stored in a reservoir, comprising the steps of:
a) providing a vessel having an interior holding a volume of nutrient in liquid medium;
b) adding a volume of bacteria to the nutrient liquid medium;
c) providing a fluid transfer system that is in fluid communication with the vessel, the transfer system including an influent flow line, a pump and an effluent flow line;
d) for a first time interval, recirculating the combination of nutrient liquid and bacteria through a flow path that begins within the vessel interior and flows from the vessel via the effluent flow line to the pump, returning to the vessel interior via the influent flow line;
e) after the first time interval, transmitting a volume of the combination of nutrient liquid medium and bacterial to the reservoir; and
f) wherein in step “d” the bacteria concentration increases to a concentrate of between about 1.0×107 to 1.0×1010 cfu (colony forming units) per milliliter (ml).

2. The method of claim 1 wherein the ratio of the volume of the reservoir or average influent flow rate to the vessel is at least 100,000-10,000,000 gallons to 1.

3. The method of claim 1 wherein the ratio of the volume of the reservoir to the vessel is between about 100,000 and 1,000,000 to 1.

4. The method of claim 1 wherein the fluid transfer system includes a vent port for aeration and further comprising aerating the nutrient liquid medium in step “d”.

5. The method of claim 1 wherein steps “b”, “c” and “e” are repeated for the same remediation of the reservoir within a time period of between about 12 to 72 hours.

6. The method of claim 1 wherein the nutrient medium includes concentrations of phosphorus and nitrogen to sustain bacterial cell division and microbial growth, the concentration of phosphorus is between about 0.5 mg/L and 10 mg/L and usually between about 1.0 mg/L and 5 mg/L, and the nitrogen concentration is between about 0.5 mg/L and 25 mg/L, more typically between about 2.5 mg/L to 15 mg/L.

7. The method of claim 1 wherein the bacteria includes strains of aerobic Bacillus and Pseudomonas species.

8. The method of claim 1 wherein in step “a” the medium includes water and varying concentrations of methanol and a nutrient.

9. The method of claim 1 wherein in step “a” the nutrient liquid medium includes water and varying concentrations of methanol.

10. The method of claim 1 wherein in step “d” the recirculating is at a flow rate of between about 50 and 90 gallons per minute.

11. The method of claim 1 wherein in step “d” recirculating is at a flow rate of between about 55 to 88 gallons per minute.

12. The method of claim 1 wherein in step “d” dissolved oxygen in the nutrient liquid is between about 0.5 mg/L and 100 mg/L.

13. The method of claim 1 wherein in step “d” the dissolved oxygen uptake rate is between about 50 and 1500 mg/L/hr.

14. The method of claim 1 wherein in step “d” recirculating is at a flow rate that does not substantially damage cells.

15. The method of claim 1 wherein the vessel has a volume of between about 25 and 500 gallons.

16. The method of claim 1 wherein the vessel has a cylindrically shaped section.

17. The method of claim 1 wherein the vessel has a conically shaped section.

18. The method of claim 1 wherein the first time interval is between about four and twenty-four hours.

19. The method of claim 1 wherein the critical cell mass concentration is more than 1.0×107 per milliliter.

20. The method of claim 1 wherein the critical cell mass concentration is more than 1.0×108 per milliliter.