Application of image analysis in microecophysiology research : methodology development.
Rehabilitation of landfill sites is important for successful land utilization. Revegetation is one key element of the process since it can overcome aesthetic problems. The inimical challenges of landfill leachate and gas are largely responsible for the difficulties associated with the revegetation of completed sites. Many components of landfill leachate can be catabolized by microbial associations thereby reducing their impacts on the environment. The importance of research on interactions between pollutants, microorganisms and soil is its applicability in environmental risk assessment and impact studies of organic pollutants which enter the soil either accidentally or intentionally. The application of image analysis with microscopy techniques to landfill soil-pollution interactions provides a means to study surface microbiology directly and to investigate microbial cells under highly controlled conditions. This research focused on the development of a method to study the real time processes of attachment, establishment, growth and division of microbial cells/associations in site covering soils. Image analysis provides a powerful tool for differential quantification of microbial number, identification of morphotypes and their respective responses to microenvironment changes. This minimal disturbance technique of examining visually complex images utilizes the spatial distributions and metabolic sensitivities of microbial species. It was, therefore, used to examine hexanoic acid catabolizing species, both free-living and in a biofilm, with respect to obviating the threat of hexanoic acid to reclamation strategies. The three sources of inoculum (soil cover, soil from the landfill base liner and municipal refuse) were compared for their ability to provide associations which catabolized the substrate rapidly. During the enrichment programme the inocula were challenged with different concentrations of hexanoic acid, a common landfill intermediate. From the rates at which the substrate was catabolized conclusions were drawn on which concentration of hexanoate facilitated the fastest enrichment. The results of initial batch culture enrichments confirmed that the soil used contained microbial associations capable of catabolizing hexanoic acid at concentrations < 50mM, a key leachate component. Exposing the landfill top soil microorganisms to a progressive increase in hexanoic acid concentration ensured that catabolic populations developed which, in situ, should reduce the phytotoxic threat to plants subsequently grown on the landfill cover. The analysis of surface colonization was simplified by examining the initial growth on newly-exposed surfaces. The microbial associations generated complex images which were visually difficult to quantify. Nevertheless, the dimensional and morphological exclusions which were incorporated in the image analysis software permitted the quantification of selected components of the associations although morphology alone was inadequate to confirm identification. The effects of increasing the dilution rate and substrate concentration on the growth of surface-attached associations in Continuous Culture Microscopy Units (CCMUs) were examined. Of the five dilution rates examined the most extensive biofilm development (9.88 jum2) during the selected time period (72h) resulted at a dilution rate of 0.5h' (at 10mM hexanoic acid). The highest growth (608 microorganisms.field"1) was recorded in the presence of 50mM hexanoic acid (D = 0.5h"1). To ensure that the different morphotypes of the associations were able to multiply under the defined conditions a detailed investigation of the component morphotypes was made. Numerically, after 60h of open culture cultivation in the presence of 50mM hexanoic acid, rods were the predominant bacterial morphotypes (43.74 field'1) in the biofilms. Both rods and cocci were distributed throughout the CCMUs whereas the less numerous fungal hyphae (0.25 field'1) were concentrated near the effluent port. The specific growth rates of the surface-attached associations and the component morphotypes were determined by area (//m2) colonized and number of microorganisms.field"' and compared to aerobic planktonic landfill associations. From area determinations ( > 0.16 h'1) and the number of microorganisms.field"1 10mM hexanoic acid was found to support the highest specific growth rate ( > 0.05 h"1) of the surfaceattached association isolated from municipal refuse. With optical density determinations, the highest specific growth rate (0.01 h'1) was recorded with 25mM hexanoic acid. The surface-attached microbial associations component species determinations by area and number showed that the hyphae had the highest specific growth rate ( > 0.11 h"1). The surface-attached microbial association specific growth rate determinations from the discriminated phase (0.023 h'1), area colonized (0.023 h"1) and number of microorganisms (0.027 h"1) calculated from the results of the component species rather than the association should give more accurate results. The specific growth rate obtained differed depending on the method of determination. Any one of these may be the "correct" answer under the cultivation conditions. Depending on the state (thickness) of the association (free-living, monolayer or thick biofilm) the different monitoring methods may be employed to determine the growth. As a consequence of the results of this study, the kinetics of microbial colonization of surfaces in situ may be subjected to the same degree of mathematical analysis as the kinetics of homogeneous cultures. This type of analysis is needed if quantitative studies of microbial growth are to be extended to surfaces in various natural and artificial environments.