Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67-85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.

Effects of di- and polysaccharide formulations and storage conditions on survival of freeze-dried Sphingobium sp.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.

A new concept for reduction of diffuse contamination by simultaneous application of pesticide and pesticide-degrading microorganisms.

Pesticide residues and their transformation products are frequently found in groundwater and surface waters. This study examined whether adding pesticide-degrading microorganisms simultaneously with the pesticide at application could significantly reduce diffuse contamination from pesticide use. Degradation of the phenoxyacetic acid herbicides MCPA (4-chloro-2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) was studied in soil microcosm experiments after simultaneous spraying of herbicide and herbicide-degrading bacteria on an agricultural soil and on a sand with low degradation potential. The latter represented pesticide use on non-agricultural soils poor in microbial activity. Degradation and possible loss of herbicidal effect were also tested in a system with plants and the amounts of bacteria needed to give satisfactory MCPA-degradation rate and the survival of degrading bacteria in formulated MCPA were determined. The results showed >80-99% degradation of 2,4-D and MCPA in soil within 1 day and >99% within 3 days after inoculation with 10(5)-10(7) herbicide-degrading bacteria g(-1) dry weight of soil. Enhanced degradation of MCPA was also obtained in the presence of winter wheat and white mustard without loss of the intended herbicidal effect on white mustard. The survival of an isolated MCPA-degrading Sphingomonas sp. in three realistic concentrations of formulated MCPA was very poor, showing that in practical applications direct contact between the microorganisms and the pesticide formulation must be precluded. The applicability and economic feasibility of the method and the information needed to obtain a useable product for field use are discussed.