Contents - Akademi Sains Malaysia

ASM Science Journal, Volume 7(1), 2013samples (0 cm – 10 cm) were obtained from the 4, 9, 14 and19 year-old forests through random sampling design. Eachsite consisted of demarcated 20 m 3 20 m area. Sampleswere collected in 3 composites, with each consisting of5 sampling points with 4 subsamples per point. Sampleswere sieved using 2 mm-sieve and diluted before plating onthree types of selective media. Soil moisture content wasdetermined gravimetrically by oven drying 10 g of freshsieved soil for at least 24 h at 105ºC. Ten g of sieved soilwas added to 95 ml of sterile 0.85% saline solution andagitated at 180 r.p.m. for 10 min prior to serial dilutions formicrobial enumeration purposes.Enumeration of Nitrogen-fixing Micro-organismsNitrogen fixing micro-organisms were grown on nitrogenfreemalate (Nfb) medium (Döbereiner & Day 1976). TheNfb plates were incubated at 30ºC for 7 days. Colonieswith a blue halo were counted in triplicates and the colonyforming unit (cfu) number was calculated.Enumeration of Phosphate Solubilizing Microorganisms(PSM)PSM enumeration technique is based on the formation ofhalos around the colonies of micro-organisms capable tosolubilize calcium phosphate (Nautiyal 1999; Gupta etal. 1994). The micro-organisms were grown in mediumcontaining insoluble phosphate (Kokal 1990; Buis 1995)and incubated at 30ºC for 12 days.Enumeration of Cellulolytic Micro-organismsThe cellulolytic micro-organisms were enumerated intriplicate by spreading plate inoculation onto cellulose-Congo red agar (Hendricks et al. 1995). Followingincubation at 30ºC for 7 days, the colonies exhibiting zonesof clearing were counted.Calculations and Statistical Analysis for Enumerationof Micro-organismsThe number of colonies per plate for the dilution givingbetween 30 and 300 colonies was averaged (Germida & deFreitas 2008). The number of cfu/g dry soil was determinedas follows:Number of=cfu/g soil(Mean plate count) 3 (Dilution factor)Dry weight soil of initial dilutionWhere,Dry weight soil = (Weight moist soil of initial dilution) 3[1– (% moisture, soil sample/100)]The counts of nitrogen fixing, phosphate solubilizingand cellulolytic micro-organisms detected by the respectivemedia were analyzed statistically using trend analysis.Statistical Analysis System version 9.1 was used for thestatistical analysis. The decision level for hypothesistesting was at p ≤ 0.05.RESULTSThe experimental data revealed the positive linear relationshipbetween age of the rehabilitated forest and microbialcounts. The adjusted R 2 was highest for the relationshipbetween forest age and cellulolytic microbial count ata value of 0.84 (Figure 1), followed by the relationshipbetween nitrogen fixing microbial count and age (R 2 = 0.83).The lowest degree of relationship was observed betweenphosphate solubilizing microbial count and forest age at R 2value of 0.78. Cellulolytic microbial count (Count C ) withforest age had a functional relationship of Count C = 25.31+ 1.94 Age, while nitrogen-fixing microbial count (Count N )with age had its functional relationship as Count N = 15.23+ 1.07 Age. Functional relationship, Count P = 6.74 + 1.11Age was used to describe phosphate-solubilizing microbialcount and forest age relationship.DISCUSSIONIt was observed that there was a general trend of increasingcellulolytic, nitrogen-fixing and phosphate solubilizingmicrobial counts over the ages of forest (Table 1 and Figure1). Many factors affect soil microbial population. It is wellknown that the interplay of varieties of factors such asphysicochemical properties of soil, temperature, vegetation,carbon availability and environmental conditions greatlyaffect soil microbial flora in terms of its numbers andactivities (Suliasih 2005; Jha et al. 1992; Setiadi 1989).Changes in the number of soil micro-organisms can affectchanges in the organic matter content of soil (Odum 1965;Zak et al. 1994) and hence when observed altogether withother ecological indicators such as biomass, and speciesdiversity measurements etc., changes in the numbersof these micro-organisms can also act as an indicator ofchanges in soil health and productivity (Jastrow & Miller1991; McBride et al. 1993).Findings of Matias et al. (2007) revealed that soilphosphate solubilizing and cellulolytic population wereinhibited by soil disturbance while the total bacterialand fungal population were not changed in the degradedsoils of iron-ore mined land. It was also reported thatmicro-organisms exhibiting nutritional variability wereless negatively impacted by lack of soil nutrition ratherthan those that need a specific substrate for their growth(Matias et al. 2007). Matias et al. (2007) stated thatmicrobial life in degraded lands such as mined lands growsonly in collaboration with the root systems of plants.Since vegetations have varying chemical composition,physiological and nutritional properties, they would be24