John R. Haines

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure.

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The methods accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

Effects of nitrogen source on crude oil biodegradation

SummaryThe effects of NH4Cl and KNO3 on biodegradation of light Arabian crude oil by an oil-degrading enrichment culture were studied in respirometers. In poorly buffered sea salts medium, the pH decreased dramatically in cultures that contained NH4Cl, but not in those supplied with KNO3. The ammonia-associated pH decline was severe enough to completely stop oil biodegradation as measured by oxygen uptake. Regular adjustment of the culture pH allowed oil biodegradation to proceed normally. A small amount of nitrate accumulated in all cultures that contained ammonia, but nitrification accounted for less than 5% of the acid that was observed. The nitrification inhibitor, nitrapyrin, had no effect on the production of nitrate or acid in ammonia-containing cultures. When the culture pH was controlled, either by regular adjustment of the culture pH or by supplying adequate buffering capacity in the growth medium, the rate and extent of oil biodegradation were similar in NH4Cl- and KNO3-containing cultures. the lag time was shorter in pH-controlled cultures supplied with ammonia than in nitrate-containing cultures.

Efficacy of commercial inocula in enhancing biodegradation of weathered crude oil contaminating a Prince William Sound beach

SummaryIn a laboratory study evaluating the effectiveness of 10 commercial products in stimulating enhanced biodegradation of Alaska North Slope crude oil, two of the products provided significantly greater alkane degradation in closed flasks than indigenous Alaskan bacterial populations supplied only with excess nutrients. These two products, which were microbial in nature, were then taken to a Prince William Sound beach to determine if similar enhancements were achieveable in the field. A randomized complete block experiment was designed in which four small plots consisting of a no-nutrient control, a mineral nutrient plot, and two plots receiving mineral nutrients plus the two products were laid out in random order on a beach in Prince William Sound that had been contaminated 16 months earlier from the Exxon Valdez spill. These four plots comprised a ‘block’ of treatments, each oil residue weight and alkane hydrocarbon profile changes. The results indicated no significant differences (P<0.05) among the four treatments in the 27-day time period of the experiment. A statistical power analysis, however, revealed that the variability in the data prevented a firm conclusion in this regard. Failure to detect significant differences was attributed not only to variability in the data but also to the highly weathered nature of the oil and the lack of sufficient time for biodegradation to take place.

Bioremediation and Biorestoration of a Crube Oil-Contaminated Freshwater Wetland on the St.Lawrence River

Biostimulation by nutrient enrichment and phytoremediation were studied for the restoration of an acutely stressed freshwater wetland experimentally exposed to crude oil. The research was carried out along the shores of the St. Lawarence River at Ste. Croix, Quebec, Canada. The research determined the effectiveness of fertilizer addition in enhancing the biodegradation rates of residual oil. It further examined the rate at which the stressed ecosystem recovered with and without the addition of inorganic fertilizers and the role of nutrients in enhancing wetland restoration in the absence of healthy wetland plants. Chemical analysis of integrated sediment core samples to the depth of oil penetration within the experimental plots indicated that addition of inorganic nutrients did not enhance the disappearance of alkanes or PAHs. In surface samples, however, hydrocarbon disappearance rates were higher when the metabolic activity of wetland plants was suppressed by the removal of emergent plant growth. These results suggest that oxygen limitation plays a major role in preventing rapid biodegradation of hydrocarbons in anoxic wetland sediment.

Biostimulation for the treatment of an oil-contaminated coastal salt marsh.

A field study was conducted on a coastal salt marsh in Nova Scotia, Canada, during the summer of 2000. The objective of the study was to assess the effectiveness of biostimulation in restoring an oil-contaminated coastal marsh dominated by Spartina alterniflora under north-temperate conditions. Three remediation treatments were tested with two additional unoiled treatments, with and without added nutrients, serving as controls. This research determined the effectiveness of nitrogen and phosphorus addition for accelerating oil disappearance, the role of nutrients in enhancing restoration in the absence of wetland plants, and the rate at which the stressed salt marsh recovered. Petroleum hydrocarbons were analyzed by gas chromatography/mass spectrometry (GC/MS). Statistically significant treatment differences were observed for alkanes but not aromatics in sediment samples. No differences were evident in above-ground vegetation samples. GC/MS-resolved alkanes and aromatics degraded substantially (>90% and >80%, respectively) after 20 weeks with no loss of TPH. Biodegradation was determined to be the main oil removal mechanism rather than physical washout.

Efficacy of commercial products in enhancing oil biodegradation in closed laboratory reactors

SummaryA laboratory screening protocol was designed and conducted to test the efficacy of eight commercial bacterial cultures and two non-bacterial products in enhancing the biodegradation of weathered Alaska North Slope crude oil in closed flasks. Three lines of evidence were used to support the decision to progress to field testing in Prince William Sound: rapid onset and high rate of oxygen uptake, substantial growth of oil degraders, and significant degradation of the aliphatic and aromatic hydrocarbon fractions of the weathered Alaska North Slope crude oil. A product had to enhance biodegradation greater than that achieved with excess mineral nutrients. Experiments were conducted in closed respirometer flasks and shake flasks, using seawater from Prince William Sound and weathered crude oil from a contaminated beach. Analysis of the data resulted in selection of two of the ten products for field testing. Both were bacterial products. Findings suggested that the indigenous Alaskan microorganisms were primarily responsible for the biodegradation in the closed flasks and respirometer vessels.

Screening of commercial inocula for efficacy in stimulating oil biodegradation in closed laboratory system

Abstract In February, 1990, the U.S. Environmental Protection Agency issued a public solicitation for proposals to the bioremediation industry on testing the efficacy of commercial microbial products for enhancing degradation of weathered Alaskan crude oil. The Agency commissioned the National Environmental Technology Applications Corporation (NETAC), a non-profit corporation dedicated to the commercialization of environmental technologies, to convene a panel of experts to review the proposals and choose those that offered the most promise for success in the field. Forty proposals were submitted, and 11 were selected for the first phase of a two-tiered testing protocol (only 10 were tested because one company did not participate). The laboratory testing consisted of electrolytic respirometers set up to measure oxygen uptake over time and shake flasks to measure oil degradation and microbial growth. If one or more products were found effective, the second tier would take place, consisting of small field plots on an actual contaminated beach in Prince William Sound in the summer of 1990. This paper discusses the first respirometric evaluations.

Protocol for testing bioremediation products against weathered Alaskan crude oil

ABSTRACT To screen various commercial microbial products for effectiveness in enhancing biodegradation of weathered Alaskan crude oil, a series of laboratory tests were conducted of 10 different products. The products were tested by electrolytic respirometry, to measure oxygen uptake over time, and in shaker flask microcosms, to measure oil degradation and microbial growth. Results supported the decision to field test two of the products.

Microbial Population Analysis as a Measure of Ecosystem Restoration

During a controlled oil spill study in a freshwater wetland, four methods were used to track changes in microbial populations in response to in situ remediation treatments, including nutrient amendments and the removal of surface vegetation. Most probable number (MPN) estimates of alkane and aromatic hydrocarbon degraders showed divergence of the alkane and aromatic degrading populations during the first summer of the experiment. Alkane degraders increased in all plots by 1.5 orders of magnitude and aromatic degraders increased in oiled plots by 3.5 orders of magnitude. Phospholipid fatty acid (PLFA) analysis of biomass and community composition showed no essential differences among treatments. Denaturing gradient gel electrophoresis (DGGE) analysis of the sediment microbial community showed some differences in specific populations of organisms with respect to oiled and unoiled plots. Some organisms were only found in the oiled plots. Sediment toxicity measured against single celled algae showed that the oiled sediments were toxic into the second year of the study, but that nutrient addition relieved the toxicity more rapidly than natural attenuation of the oil.

TESTING THE EFFICACY OF OIL SPILL BIOREMEDIATION PRODUCTS

ABSTRACT Ten bioremediation products were tested in laboratory respirometers for their ability to enhance the biodegradation of artificially weathered Alaska North Slope crude oil compared to natur...