Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of...
Reexamination Certificate
2000-05-05
2002-12-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
C435S260000, C435S262000, C435S262500, C435S267000, C435S268000, C435S289100, C435S307100, C435S801000, C435S821000
Reexamination Certificate
active
06489156
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the isolation and use of bacterial strains for odor remediation and fermentation in anaerobic livestock waste lagoons. In particular, the invention relates to the isolation of a biologically pure culture of a Rhodobacter PS9 (“RPS9”) bacterial strain and methods of using the bacterial strain to reduce the concentration of malodorous volatile organic compounds in anaerobic livestock waste lagoons and for biomass production from genetically reconstituted RPS9 cells.
REFERENCES
Several publications are referenced herein. Full citations for these publications are provided below. The disclosures of these publications are hereby incorporated herein by reference in their entirety, unless otherwise noted.
BACKGROUND OF THE INVENTION
Livestock waste lagoons are large reservoirs used for the storage and treatment of livestock waste and runoff. In order to operate efficiently, the waste must be loaded at an appropriate rate and maintained at a minimum volume. Such lagoons may be aerobic, anaerobic or facultative. While aerobic waste lagoons control sludge and odor better than anaerobic lagoons, they require the use of mechanical aerators to ensure adequate oxygen. In contrast, anaerobic lagoons can treat high waste load rates, do not require aeration, and thus operate at a much lower cost.
Anaerobic lagoons are often associated with noxious odors causing environmental problems associated with their use. In addition, the proximity of waste lagoons to populated areas exposes waste lagoon operators to nuisance lawsuits and potential liability. To help reduce problems associated with noxious odors, a two-stage anaerobic lagoon may be employed, wherein treated waste from a first cell is transferred to a second adjacent cell. However, operation of a two-stage lagoon is expensive. The main source of odors associated with anaerobic a waste lagoons results from the anaerobic digestion of complex, volatile, organic compounds resulting in the production of malodorous volatile organic and inorganic compounds.
U.S. Pat. No. 5,861,096 is directed to reducing odors from wastewater by chemical treatment. According to the patent, the most common source of odors in wastewater is reduced sulfur compounds resulting from anaerobic bacterial decomposition of biodegradable organic matter in the presence of sulfates. The patent refers to spraying an aqueous solution of a chlorine compound over the surface of a wastewater lagoon to react with the sulfides emanating from the surface. The spray may be provided through spray nozzles using a pipe distribution system and must cover at least 1,000 square feet.
U.S. Pat. No. 5,744,041 is directed to adding undefined cultures of anaerobic bacteria to wastewater to reduce the biological oxygen demand (BOD) of the wastewater. A portion of the treated wastewater with a reduced BOD is mixed with aerobic bacteria to form a liquor comprising water and aerobic organisms. The liquor is subjected to microzone treatment to remove a portion of the aerobic microorganisms. “Genetically engineered” or “genetically reconstituted” organisms are often used for large-scale biomass production. Typically, a gene encoding a protein of interest is cloned into a bacteria using a vector system. The bacteria are grown in a large-scale fermentation process and induced to express the protein of interest which is then isolated from the bacterial cells. Significant costs are associated with establishing and maintaining particular bacterial growth conditions, including providing the appropriate growth media, temperature, pH, and oxygen level. Most plasmid vector systems rely upon use of antibiotic selection genes to maintain the presence of the vector in the cell. Furthermore, vector systems typically require the addition of exogenous compounds to induce expression of genes under the control of promoter sequences.
What is needed are new methods of reducing the odors associated with livestock waste lagoons in a reproducible, cost-effective and environmentally friendly manner. Systems and methods for large-scale biomass production are also needed that rely on naturally-occurring conditions and do not require the addition of antibiotics or other exogenous compounds.
SUMMARY OF THE INVENTION
This invention provides a newly isolated Rhodobacter PS9 strain and methods of using such strain to reduce the odor associated with livestock waste lagoons. Also provided herein are methods of using genetically reconstituted Rhodobacter PS9 cells for large-scale biomass/enzyme production.
The present inventors have identified RPS9 as the major photosynthetic bacterium in certain anaerobic livestock waste lagoons. In these photosynthetic lagoons, this bacterium constitutes 40 to 60% of the total microbial populations. The present invention provides a newly isolated photosynthetic bacterial strain, RPS9. Cultures of RPS9 are used, according to a preferred embodiment of the invention, to inoculate malodorous livestock waste lagoons and reduce odor through degradation of various volatile organic compounds.
In one preferred embodiment of the invention, a biologically pure culture of bacteria having some or all of the identifying characteristics of RPS9 is provided. In this embodiment, the identifying characteristics of RPS9 may include a bacteriochlorophyll &agr; (“Bchl”) profile, a cell extract absorption spectra, a MIDI-FAME (Microbial Identification System-Fatty Acid Methyl Ester) profile, and a species specific rRNA profile. The biologically pure culture bacteria is preferably capable of degrading malodorous volatile organic compounds such as fatty acids (e.g., butanoic acid, pentanoic acid, hexanoic acid, and heptanoic acid) and aromatic compounds (e.g., phenol, para-cresol, and 3-methyl-indole).
In another preferred embodiment, a method of inducing the growth of RPS9 and variants thereof in a livestock waste lagoon is provided by controlling lagoon conditions such as pH, temperature, and organic load. Growth is enhanced by adjusting the temperature to at least at about 22° C. or above, the pH to about pH 6.9 to about 7.8, and the organic load to a protein concentration of about 1 g per liter and, preferably, a dry weight below about 4 g per liter.
In another preferred embodiment, an inoculum comprising a biologically pure culture of RPS9 or variant thereof and a suitable carrier is provided. The inoculum is used to inoculate a livestock waste lagoon. The inoculum is typically used to inoculate a livestock waste lagoon by providing from about 1 ml to about 5 ml of the bacterium (at a concentration of about 2 mg cell protein/ml) to the lagoon by any suitable method, preferably after adjusting the temperature, pH and organic load of the livestock waste lagoon.
In a further preferred embodiment, a method of genetically reconstituting a RPS9 strain is provided by introducing a vector comprising a gene encoding a protein product, a promoter to control expression of the protein product, and a selection gene. Preferably, the vector system does not require the continual presence of antibiotics to maintain the vector in the RPS9 strain. The promoter preferably does not require the addition of a substrate to induce expression of the protein product. The selection gene is preferably a gene essential to the survival of the RPS9 strain. A particularly preferred selection gene is the ffh gene. Reece, K. R and Phillips, G. J. (1995); Phillips, G. J. and T. J. Silhavy (1992). The gene may be inserted into the vector using any suitable recombinant DNA technique or method. The vector may be introduced into the RPS9 strain using any suitable technique or method.
In a yet another preferred embodiment, a fermentation system and method for producing a protein is provided. A genetically reconstituted RPS9 strain or variant thereof is grown in a livestock waste lagoon. The RPS9 cells are preferably separated from the liquid waste and induced to express a protein product in an inducing media. The protein product is preferably separated from the induced RPS9 cells. The fermentation
DiSpirito Alan A.
Do Young S.
Phillips Gregory J.
Zahn James A.
Iowa State University & Research Foundation, Inc.
Prouty Rebecca E.
Rao Manjunath
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