Biodegradation of spilled diesel fuel in agricultural soil: effect of humates, zeolite, and bioaugmentation.

Possible enhancement of biodegradation of petroleum hydrocarbons in agricultural soil after tank truck accident (~5000 mg/kg dry soil initial concentration) by bioaugmentation of diesel degrading Pseudomonas fluorescens strain and addition of abiotic additives (humates, zeolite) was studied in a 9-month pot experiment. The biodegradation process was followed by means of analytical parameters (hydrocarbon index expressed as content of C10-C40 aliphatic hydrocarbons, ratio pristane/C17, and total organic carbon content) and characterization of soil microbial community (content of phospholipid fatty acids (PLFA) as an indicator of living microbial biomass, respiration, and dehydrogenase activity). The concentration of petroleum hydrocarbons (C10-C40) was successfully reduced by ~60% in all 15 experiment variants. The bioaugmentation resulted in faster hydrocarbon elimination. On the contrary, the addition of humates and zeolite caused only a negligible increase in the degradation rate. These factors, however, affected significantly the amount of PLFA. The humates caused significantly faster increase of the total PLFA suggesting improvement of the soil microenvironment. Zeolite caused significantly slower increase of the total PLFA; nevertheless it aided in homogenization of the soil. Comparison of microbial activities and total PLFA revealed that only a small fraction of autochthonous microbes took part in the biodegradation which confirms that bioaugmentation was the most important treatment.

Determination of OH groups in humic acids using methylation with dimethylsulfate.

The methylation of humic acids (HA) with dimethylsulfate in acetone and methanol followed by the iodometric determination of the methoxy groups (Zeisel reaction) were applied to determine the contents of -OH groups in solid samples of HA of different origins. For the coal- and peat-derived HA samples, the contents of -OH groups determined after methylation in acetone ranged from 6.6 to 8.7 mmol/g, whereas the contents of -OH groups determined after methylation in methanol ranged from 4.0 to 5.0 mmol/g. These differences may be related to the content of carboxylic groups in the HA molecule that were not methylated in methanol, as confirmed by a comparison with results of conventional titrimetric determinations. Observed differences were interpreted as results of different polarity of both solvents and alkalinity of the reaction mixture during the methylation. The contents of alcoholic groups as well as some other minor -OH groups can be estimated using the -OH group contents obtained after methylation in both solvents together with the results of the conventional determinations of acidic functional groups. A repeatability of the -OH groups determination as estimated from a series of triplicate analyses of different HA samples (n=7) was in range of 0.15-0.73 mmol/g and 0.08-1.06 mmol/g (standard deviations) for methylation in acetone and methanol, respectively. Thus, the average repeatability of the -OH groups determination was estimated to be 0.38 and 0.50 mmol/g for methylation in acetone and methanol, respectively.