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i — i� wow Effects of Nutrient Supplementation: Nutrient evaluations are performed to determine if inor- ganic nutrient supplementation (i.e., nitrate) will result in enhanced microbial growth. Site-specific samples are enriched with varying concentrations of nutrients, the types) and concentration(s) based on which nutrients were deficient as determined by the results of baseline physicochemical analyses. Following an adaptation (growth) period, heterotrophic and selective microbial enumera- tions are performed to comparatively evaluate microbial growth with respect to nutrient supple- mentation. Based on these results, it can be determined whether low nutrient levels may be a limiting factor at the site and, if so, what corrections, such as an increase in nitrate levels, can be made that would result in an environment capable of promoting microbial growth and biodegrada- tion. - Effects of pH Variations: Evaluations on the effects of pH on microbial growth are performed to determine if ambient (site-specific) pH is a limiting growth factor within the site matrix. Micro- bial enumerations are performed on site-specific samples, typically at pH values one to two units lower and higher than ambient pH. By comparing microbial growth patterns at varying pH values (including ambient pH), the results can be used to determine if the ambient pH is conducive to promoting microbial growth and biodegradation, and, if so, whether significant pH fluctuations could adversely affect the biodegradative process. - Effects of Oxygen Supplementation: Oxygen deficiency in in situ environments is a well documented limiting factor to aerobic biodegradation processes. The purpose of evaluating the effects of oxygen supplementation at varying concentrations is to determine the level of oxygen required to promote microbial growth and biodegradation within the contaminated matrix. Based on dissolved oxygen (DO) measurements obtained from field analyses, aliquots of site-specific samples are enriched with varying concentrations of oxygen (in the form of hydrogen peroxide). Following an adaptation period, as a measure of microbial growth, turbidity determinations are performed utilizing a spectrophotometer. Comparative observations are made to determine if the site-specific concentration of oxygen is acting as a growth-limiting factor. - Effects of Temperature Variations: Evaluation of the effects of variations in temperature are performed to determine the temperature range at which achievable and/or optimal microbial growth would occur. Heterotrophic and selective microbial plate count assays are performed at low (4°C), moderate (27-30°C) and high (37-40°C) temperatures and the differences in microbial growth are observed. The results are used to determine if significant temperature fluctuations, such as cold, snowy conditions, within the site matrix during prospective biotreatment could result in limiting microbial growth and biodegradative efficiency. This analysis can also assist in the development of thermophilic (heat-loving) or psychrophilic (cold-loving) bacteria that can be used preferentially for applicable sites. Effects of Contaminant Level Variations: Many sites vary in the concentration of contami- nant present at different sampling points; therefore, it is important to determine the upper (toxic) and lower (threshold) concentration limits for a bioremedial system at any one point. The purpose of these evaluations is to determine the contaminant concentration range at which indigenous mi- crobial growth will occur. Site-specific samples are plated on selective culture media supplemented with varying concentrations of contaminant. Following an adaptation period, microbial enumera- tions are performed and growth patterns compared. The results can provide information on whether and to what degree biodegradative activity will occur at sampling points where contamination levels are of concern (too low/high). 6