I. INTRODUCTION going on since life began on the planet earth, Ith sure eventually has healed itself. In today’s rapidly m . But there are no such things as instant wi 0 is the case with environmental bioremediation. First we nocd ¢ on) in the environment, and then the adaptation of existing environment. Once these conditions exist, the bacteria begin the elaborate process o fhem ds an energy source and in the proce é Sng this natural bioremediation process, nature catcrs to the nutritional and physiological needs of the bacteria. This natural process is slow, taking many years compared with mari-assisted program that will speed up the environmental bioremediation. This is accomplished by increasing up front the number of bacteria and by paying attention to the practical considerations at the contaminated spill site, e.g., pumping and collécting excess petroleum, and providing for the growth needs of the petroleum-degrading microbes ontami of that contaminant. Du il, PRACTICAL CONSIDERATIONS Many of the practical aspects used in. bioreméstiation have been discussed in conversation or used in the field and need to be reportec’ in some logical sequence. The process of bioremediation generally can be accomplished in four basic steps.! The first step surveys the problem and inchides determination of the type of containination, e.g., JP-4 Jet fuel, the extent of the contamination, the quantity of contaminated soil to be remediated, the properties of the soil, and identification of the natural fuel-degrsding bacteria at the site. Bacteria isolated from the fuel spill site are identified for use in the bioremediation process. The second step involves increasing'the number of bacteria by a proces known as fermentation to a biomass (total mass of living microorganisms in a given volume). It is this biomass (large number of bacteria) that a 3940 Fletcher will degrade the contaminant in the soil. Step 3 is the application of the biomass to the petroleum hydrocarbon contaminated soil by gravity drip, injection, or spray techniques. Step 4 involves, monitoring and maintaining ideal growth conditions for the bacteria by providing nutrients, oxygen, moisture, and an optimum acid-base balance. In more detail, some of these practical s are given in what follows. A. Microbe Isolation and Identification Bacteria, molds, or yeast could be selected! for an environmental bioremediation program, but this discussion shall concentrate on facultative anaerobes or aerobic bacteria, which in most ses efficiently degrade surface petroleum spills. These bacteria are often of the genera Pseudomonas, Acinetobacter, Citrobacter, Flavobacterium, or Chromobacter. An old petro- Jeum spill site usually has many bacteria that are active in degrading petroleum. Bacteria at a fresh spill site are undergoing transition with petroleum-sensitive bacteria dying off (bactericidal effect), or their multiplication being inhibited (bacteriostatic effect). Some bacteria, however, ijusting to the contaminant by using the petroleum as a carboh source for their nutritional Js, These bacteria are selected for isolation, identification, and determination of petroleum- ading ability. The isolation techniques involve vacious bacteriological procedures depending on the investigator or the company that is attempting to isolate and identify the oil-degrading bacteria, In our laboratory we bave successfully employed the following techniques. 1. Methods and Materials for Bacteria Isolation Several soil samples are collected (approximately 100 g each) from the center and perimeter of the contamination site. These individual samples are placed in sterile containers and 0.1 g of each inoculated into 9.9 mi of sterile trypticase soy broth, thoroughly mixed (vortexed) for 60 s, and 0.01 ml used to inoculate a minimal nitrogen medium (Table 1). This nitrogen medium was adjusted to pH 7.0 and autoclaved at 15 psi for 15 min before being dispensed in 16 x 125 mm screw cap tubes. Each tube of this nitrogen medium is overlayed with | ml of the petroleum contaminant before it is inoculated with the soil specimen, ‘The soil specimen (0.01.m) is inoculated through the petroleum overlay into the nitrogen medium, (Fig. 1). The soil-inoculated-nitrogen medium is incubated at 24°C (75.2°F) for 48 hr. The medium provides the nitrogen source and the petroleum overlay provides the carbon source for the bacteria. The bacteria that multiply in this procedure will be petroleum degrading. After the 48 hr incubation ‘period the bacteria from the petroleum-overlay-nitrogen medium are streaked on a trypticase soy agar plate and incubated for 2 to 3 days at 24°C (Fig. 2). The ial colonies are isolated from these plates and inoculated into fresh petroleum-overlay-ni- bacte Table 1 A Nitrogen Base Medium for Isolation of Soil Microorganisms (NHS, os NaNO, ose CaCl, 0.02 g MgSO, O28 KH;PO, 10g Nal!sPO, » H;0 10g Ags 19.08 Tap HzO 1000 ml pI adjusted 10 7 FigureNitrogen Medium ps broth Figure 1 1 trogen medium to provide pure bacterial cultures. These pure cultures are identified as to genus and species and evaluated for their ability to degrade the petroleum contaminant 2. Bacterial Identification The bacteria isolated is identified by standard microbial procedures? and cli procedures. These techniques are suitable for the small and large microbiology laboratori x method that is accurate and rapid is that of gas chromatography, which would provide identification of the bacteria through their fatty acid analysis.‘ This technology can be found in the large well-equipped university or industrial laboratory ation ith fhoculating Loop: Fist Stock Second Streak ‘Individual Colonies Third Streak Bacterial Growth, Figure 2 Streak plate methods for isolation of pure culture of bacteria using a trypticase soy agar.