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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
A. Tang et al.: Cellulolytic, Nitrogen-fixing and PSM as Biological Indicators of Forest Soil Rehabilitation70Microbial counts (10 4 cfu/g dry soil)605040302010Count C = 25.31+1.94 AgeR 2 = 0.84*n = 4Count N = 15.23+1.07 AgeR 2 = 0.83*n = 4Count P = 6.74+1.11 AgeR 2 = 0.78*n = 4CellulolyticN-fixingP-solubilizing00 48 12 16Rehabilitated forest age (years)20Figure 1. Relationship between cellulolytic, nitrogen fixing and phosphate solubilizing microbialcounts and age of the rehabilitated forest.Table 1. Soil microbial counts in forests of different age.Microbial groupMicrobial count (310 4 cfu/g dry soil) of selected forest age (Years)4 9 14 19Cellulolytic 30.97 ± 0.81 45.18 ± 4.08 54.00 ± 6.11 60.40 ± 6.79N-fixing 18.66 ± 1.12 25.59 ± 2.31 31.30 ± 0.70 34.59 ± 5.22P-solubilizing 9.58 ± 2.38 17.97 ± 4.31 24.52 ± 3.77 25.87 ± 1.11drawn by specific groups or species or organisms, whichmay be beneficial (Katznelson et al. 1948).The ability of micro-organisms to react quickly tochanges enables their rapid adaptation to environmentalconditions (Nielsen & Winding 2002). Thus, microbialanalyses resulted from the adaptation allow fordiscrimination in soil health assessment, and resultingaltered microbial populations and activities may thereforeprovide an excellent indicator of change in soil health(Kennedy & Papendick 1995; Pankhurst et al. 1995). Whilestudies have indicated that free-living nitrogen-fixingmicro-organisms may be suitable as biological indicatorsof heavy metal toxicity in soil in terms of their enzymaticactivities (Martensson & Witter 1990; Brookes et al. 1986),the results obtained in this studies via microbial populationsmight also prove some potentials of nitrogen-fixing microorganismsas soil health indicator in rehabilitated forest.CONCLUSIONThe soil microbial counts of cellulolytic, nitrogen-fixingand phosphate solubilizing microbes increased withincreasing age of the rehabilitated forest. Among thethree microbial populations, cellulolytic microbes haddemonstrated the highest potential which could be used asbiological indicator in regards to the rehabilitation degreeor soil health of rehabilitated forest.ACKNOWLEDGEMENTSThis research is funded by the Fundamental Research GrantScheme (FRGS; 07-04-10-908FR) under the Ministry ofHigher Education, <strong>Malaysia</strong>.Date of submission: May 2011Date of acceptance: January 2013REFERENCESAzani, AM, Majid, NM & Meguro, S 2001, ‘Rehabilitationof tropical rainforests based on indigenous species fordegraded areas in Sarawak, <strong>Malaysia</strong>’, eds S Kobayashi,JW Turnbull, T Toma, T Mori & NM Majid, in Rehabilitationof Degraded Tropical Forest Ecosystems: WorkshopProceedings, Bogor, Indonesia, 2–4 Nov. 1999, Center for25
Page 1 and 2: ContentsASM Sc. J.Volume 7(1), 2013
Page 3 and 4: INTERNATIONAL ADVISORY BOARDAhmed Z
Page 5: The Academy of SciencesMalaysia (AS
Page 8 and 9: NEWS FOCUSBio-Jet Fuel — Challeng
Page 10 and 11: ASM Science Journal, Volume 7(1), 2
Page 12 and 13: NitrogengasAcetylene gasFume hoodGa
Page 18 and 19: ControllerN 2N 2ValveValvePreheater
Page 26 and 27: ASM Sci. J., 7(1), 18-22Electron-ph
Page 28 and 29: v I= v MthASM Science Journal, Volu
Page 40 and 41: 4.2 × 10 -5Magnetic energy (Em) Ma
Page 42 and 43: 2.0 × 10 -5Inductance (L)Magnetic
Page 46 and 47: addition, examination of the freque
Page 48 and 49: equipped with a computer image anal
Page 50 and 51: Storage modulus (Pa)1.80E+101.60E+1
Page 52 and 53: (a)(b)(c)Figure 6. Micrographs: (a)
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Page 62 and 63: 3.02.53.02.02.5CB10TSSAlbipoxTimber
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Climate Change — Environment andI
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