Method for preservation of marine microorganisms

Details Method for preservation of marine microorganisms Method for preservation of marine microorganisms

1999-11-12

2002-04-23

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Preserving or maintaining micro-organism

C435S307100, C435S253600

Reexamination Certificate

active

06376229

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a the preservation of microorganisms, and, more particularly to a method for preserving oceanic microorganisms in an aqueous solution within sealed packaging providing conditions favorable for survival for periods in excess of twelve (12) months. One aspect of the invention relates to a method for packaging marine substrate material, such as sand, aragonite, coral rock and crushed coral in an aqueous solution contained within packaging, suitable for retail display and sale, wherein a suitable environment is present so that marine microorganisms remain in biofilms attached to the surface of the material for extended periods of time. A further aspect of the invention relates to the discovery of a nutrient rich seawater enrichment solution containing vitamins, organics stock, trace metals, and NaNO
3
and NaH
2
PO
4
H
2
O for further extending the period of time wherein the organisms remain bio-active.
The invention provides for the effective harvesting, packaging, transport, bio-active storage, and retail sale of aquarium substrate material containing live microorganisms, such as bacteria useful in oxidizing ammonia in aquariums, whereby the microorganisms remain biologically viable for extended periods of time in excess of twelve (12) months thereby maintaining a stable and healthy aquarium environment.
2. Description of Related Art
Aquariums have experienced a boom in popularity in recent years. Many saltwater aquariums include a diverse mix of tropical fish, live coral formations, and other exotic marine life. Saltwater marine organisms are directly affected by the chemical, biological, and physical characteristics of their environment. A number of environmental factors are critical to maintaining the delicate balance required for a healthy aquarium environment. Factors such as water temperature, pH level, lighting conditions, and complex chemical balances must constantly be maintained and monitored. The introduction of fish and other marine animals into an aquarium causes a series of chemical changes often resulting in chemical imbalances that are not conducive to aquatic life. It is therfor crucial to maintain a high level of water quality.
The initial set up of a marine aquarium typically requires a conditioning period that can take up to six (6) weeks depending upon the aquarium conditions and temperature. During the conditioning period the chemical composition of the water undergoes a series of changes and waste products can quickly build-up to levels that are toxic to aquarium life. The introduction of fish, plants, and food into an aquarium begins a natural process often referred to a “biochemical cycling”.
A significant change in the chemical composition of the water involves the accumulation of ammonia The process begins when fish and invertebrates excrete waste. The excreted waste increases the amount of ammonia present in the water as a result of decaying food and organic compounds. Harmful ammonia and nitrite are constantly converted into less harmful nitrates, which in turn is used by plants and algae for food. Aquariums are full of both autotrophic and heterotrophic bacteria that attach, grow, and form biofilms where the bacteria convert toxic nitrogeneous compounds and ammonia into harmless products. Nitrobacter and Nitrosomonas are examples of autotrophic bacteria that use oxygen to oxidize ammonia (NH
4
) to nitrite (NO
2
) and Nitrate (NO
3
).
Ammonia is a toxic waste product which, if unchecked, can accumulate and cause injury or death to aquarium inhabitants. In fact, the presence of ammonia in aquarium water is the number one cause of death in aquarium fish. The primary sources of ammonia are decaying organic material (such as uneaten food) and waste excreted by fish, other animals and organisms. An ammonia level as low as 0.5 parts-per-million (PM) creates stress in fish and compromises the natural immune systems of fish and other aquarium inhabitants. An ammonia level of 2 PPM has been found to cause the natural immune system of the fish and other aquarium inhabitants to fail or otherwise cease fuctioning. Accordingly, maintaining ammonia levels is critical to the health of the aquarium habitat.
The accumulation of ammonia is often caused by the lack of sufficient numbers of Nitrosomonas. Nitrosomonas is a genus of bacteria in aquaria that oxidize ammonia thereby regulating the ammonia level. Nitrosomonas, and other ammonia oxidizing bacteria, are found in natural abundance in marine materials, such as sand, aragonite, and crushed coral, harvested from the ocean floor. Nature provides many types of bacteria that, in the presence of oxygen, carry out the oxidation of ammonia to nitrites and eventually to nitrates in a process known as nitrification. It has been found that such bacteria settle on marine materials, such as aragonite (reef sand), and eventually form a biofilm. Marine nitrifying bacteria in the biofilm oxidize ammonia to nitrite, and nitrite to nitrate. Accordingly, these natural marine materials provide a natural source of ammonia oxidizing bacteria for use in maintaining ammonia levels in aquarium environments. Nitrate not utilized by plants is removed by other bacteria in the absence of oxygen (the anaerobic environment found in the lower levels of the sediment) in a process called denitrification.
While marine nitrifying bacteria are found in abundance in natural materials, such as aragonite harvested from the ocean floor, it has been found that there are generally three conditions that are required to maintain the nitrification process. These conditions are: (1) a surface upon which bacteria can attach, grow, and from a biofilm; (2) ammonia to start the process; and (3) an aerobic environment. The absence of any of the above-referenced conditions will either prevent of delay the nitrification process.
The initial set-up of aquariums presents unique biochemical circumstances that must be addressed in order to produce and maintain a healthy environment for marine life. The initial cycling of organic compounds in an aquarium started with dry sand or gravel often takes a period of several weeks during which an ammonia source (often only one or two small fish) provides an environment wherein beneficial bacteria to establish and begin to flourish eventually forming a biofilm. It has been found that the long initial cycling period realized when starting an aquarium with dry sand or gravel results from the time required for bacteria to attach, grow and form a biofilm on the previously dry, and organically inactive, sand and gravel. It has been shown that the initial cycling period can be substantially reduced by the introduction of bacteria rich “wet” sand and gravel that has been recently harvested from the ocean and thus contains an abundance of bacterial biofilm. Marine sand and gravel harvested from the ocean or riverbeds contain both autotrophic and heterotroplic bacteria in their natural state (i.e. established biofilms on the sand particles), each of which facilitate the rapid cycling of an aquarium. Accordingly, there exists a need for a method of harvesting and packaging marine materials such as aragonite reef sand, gravel, crushed coral and the like, such that the bacteria remain metabolically and physiologically active for extended periods of time in excess of twelve (12) months in retail packaging at room temperature.
It has proven difficult, however, to maintain ammonia oxidizing bacteria and other useful bacteria in a biologically active state during the extended period beginning with the harvesting of the material and ending with the purchase by a consumer and delivery into an aquarium; a time period often reaching up to six (6) months or more. The difficulty is increased where the harvested materials must be stored for extended periods in retail packaging at room temperatures.
The background art reveals several references directed to preserving bacteria and the lik

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