Application of image analysis in microecophysiology research : methodology development.
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Date
1998
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Abstract
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.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.
Keywords
Sanitary landfills--Environmental aspects., Waste disposal in the ground--Microbiology., Ecophysiology--Methodology., Image analysis., Theses--Microbiology.