Studies on the enhanced antagonism of Trichoderma harzianum biofilms for water treatment using slow sand filters

Abstract

Bacteri a and fungi in the natural environment grow aggregated with each other, with solid surfaces, and at gas-liquid interfaces. There is a growing appreciation that, although clearly worthwhile, stud ies of standard planktonic cultures provide us with a biased view of microbial life (Parsek et.a/2004) Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behavior which is different to that in submerged culture; thus, they can be considered as biofilm forming organisms according to this concept (Akao et.a/., 2002; Biesebeke et.al., 2002) Uncertain ramifications of fungi in potable water have led to a limited number of investigations which show that fungi are present in a significant proportion of tap water samples; however, species abundance and diversity are extremely variable (Hinzelin et.al 1985, Nagy et.al 1982, Neimi et.al 1982, Rozenzweig et.al 1986) Fungal biofilm kinetic models need to be developed yet since they have only recently been realized as true biofilms instead of simple cell immobilization systems (Keshavarz et.al., 1990; Pakula and Freeman, 1996). In the present study, a biocontrol fungi which was previously considered to be completely terrestrial, was grown and incorporated into biofilms on a laboratory scale sand filtration assembly. Although Trichoderma sp. have been found in numerous water distribution systems, processing plants and municipal supply lines, this is one of the first studies in seeing its role in chemical and microbial removal in submerged conditions and therefore its ability to "purify" water. In the present research Trichoderma halZianum was isolated from soil in Punjab, this stra in was a known bioantagonist. Its antagonistic capabilities were stud ied in the lab against Fusarium oxysporum. , Aspergillus niger., Salmoel/a typhi, Escherichia coli, Pseudomanas aeroginosa, Pseudomonas putida and Enterobacterer aerogens. It was shown to be highly effective with an efficiency rate of upto 96% removal of pathogens. This strain was then over a period of two months adapted to grow in water with minimal media, it adapted and remained viable upto two years of this study. Spore density was reduced by 60 %, there was almost complete reduction of 1 Abstract pigmentation when grown submerged in pure tap water, but green pigmentation did occur when grown in solid state on sand columns under water. Growth rate was slowed by upto 40 %, mycelia were much weaker and thinner. The fungus incorporated well onto cellulose discs and grew with bacteria, still allowing bacterial film to form with it, although this film had 18% less bacteria. That may be due to competition and lack of space more than as a result of Trichoderma antagonism. Biofim development was studied by carbohydrate production, fungal biofilms produced upto 0.6 0.55 mg/ml and in washings of planktonic cells upto 0.4 mg/ml in 10 days. Overall there was 30% greater reduction of fungal counts and 6% reduction in bacterial counts in the biofilm due to the presence of Trichoderma. Chemically Tricho.derma incorporated biofilms were better for the removal of metals such as chromium (7%) and iron (40%), salts such as nitrates (5%), Chlorides (1 %) and sulfates upto 12 %. Trichoderma was also seen to be more successful in Colour (4%), Turbidity (3%), Total dissolved solids (2%) without any significant alteration in pH . This study concluded that bioantagonist microorganisms are capable of adapting and adjusting to extreme variations in environment. Trichoderma harzianum does not loose completely its biocontrol ability even at high water potentials. It incorporated itself within the living system of a biofilm, there it utilized space and nutrients to its best advantage in a highly competitive manner and exerted its dominance over other organisms. This ability can be extremely useful if manipulated and taken advantage of in wastewater, drinking water and recreational water purification. Further studies are required to¥ test the systems in field scale and industrial levels, it is expected that presently the results are indicative of a biofilm radius of only 3 inches, this if taken at a greater and deeper level would show even better results of the potential of Trichoderma in exerting its biocontrol in aquatic microbial communities.