Isolation and Purification of Bacterial Strains from Treatment Plants for Effective and Efficient Bioconversion of Domestic Wastewater Sludge

Forty six bacterial strains were isolated from nine different sources in four treatment plants namely Indah Water Konsortium (IWK) sewage treatment plant, International Islamic University Malaysia (IIUM) treatment plant-1,-2 and –3 to evaluate the bioconversion process in terms of efficient biodegradation and bioseparation. The bacterial strains isolated were found to be 52.2% (24 isolates) and 47.8% (22 isolates) in the IWK and IIUM treatment plants respectively. The results showed that the higher microbial population (9-10x10 cfu mL ) was observed in the secondary clarifier of IWK treatment plant. Only the gram-staining identification was done in the strains isolated from IWK treatment plant not to be determined from IIUM. Among the isolates from IWK, 10 isolates of gram-positive bacillus (GPB) and gram-positive cocci (GPC), 10 isolates of gram-negative bacillus (GNB) and rest were both or undetermined. Gram-negative cocci (GNC) were not found in the isolates from IWK.


INTRODUCTION
Sewage treatment plants are artificial ecosystems in which the biodegradation of organic pollutants is carried out mainly by bacterial communities and producing excess sludge. This excess sludge presents serious disposal problems due to its huge quantity and toxicity. In Malaysia, about 3.8 million cubic meters of domestic wastewater sludge is produced annually by Indah Water Konsortium (IWK) and its management cost is estimated to RM 1 billion [1] . Effective and efficient developed bioconversion process through potential microbes in terms of biodegradation and bioseparations as well as the environmentally friendly are the main concern of IWK.
To achieve the goal, the effective bioconversion process is being considered to treat the wastewater sludge through potential microorganisms involved. In this process, the potential bacterial strains isolated from different wastewater treatment plants are involved to degrade the dissolved and suspended organic substances in sludge and enhance the biodegradation, biosolids reduction and bioseparation processes. Among microorganisms participating in activated sludge communities bacterial isolates have been attracting increasing attention in terms of faster growth, high resistance to contaminate and adaptation compared to other organisms from biotechnological point of view.
In fact bacteria of this consortia are known to be involved in biodegradation of a number of different pollutants and processes such as waste materials [2] , denitrification [3] , wastewater treatment [4] , biphenyl (BP) and chlorinated BPs [5] , phenol [6] crude oil and lubricant [7,8] . Therefore the present study was undertaken to isolate, purify and identify (gramstaining) the bacterial strains for effective bioconversion of wastewater sludge through biodegradation and bioseparation under natural conditions. Isolation and purification of bacteria: The brain heart agar (BHA) medium was used to isolate the bacterial strains from the samples. The cell counting technique in agar plates was followed to determine the population density for each sample. A series of dilutions were made to reduce the cells in the samples. One ml of diluted sample was spreaded onto the surface of BHA medium in the petri dishes and incubated at 37 0 C and allowed to grow for 24 h. Single developed colony was picked on the BHA plates and subcultured to purification. Pure bacterial strains were obtained after successive transfer of individual colony in BHA plates and incubated for 24 h at 37 0 C temperature. A partial identification only the gram staining was observed in the selected isolates.

RESULTS
A total of 46 bacterial strains were isolated from nine different sources in four treatment plants ( Table 1). The bacterial community in the brain heart agar (BHA) plates from different sources in treatment plants for isolation is shown in Fig. 1. The single bacterial colony was cultured from microbial community in BHA plate. Among them 24 strains were isolated from IWK treatment plant and 22 strains from IIUM treatment plants (three plants). The distribution of isolated bacterial strains was 52.2% and 47.8% (TP-1: 13%, TP-2: 19.6%, TP-3: 15.2%) in the IWK and IIUM treatment plants, respectively. The maximum population density (9-10x10 4 cfu mL 1 ) of bacterial communities was counted in the influent of secondary clarifier of IWK treatment plant followed by the IIUM treatment plant-3 (6-7x10 4 cfu mL 1 ), -2 (2.5-3x10 4 cfu mL 1 ) and -1 (2.2-2.5x10 4 cfu mL 1 ), respectively. The lowest density (4-5x10 3 cfu mL 1 ) was found in the effluent sample in IWK plant. The representative strains of bacterial pure culture isolated from different sources in four treatment plants after 24 hrs of incubation at 37 0 C are shown in Fig. 2. Most of the colony pigments in BHA media were appeared in light creamy and some isolates were white and yellow in the same media.   IWK1001  IWK1002  IWK1003  IWK1004  IWK1005  IWK1006  IWK1007  IWK1008  IWK1009  IWK10010 9-10x10 4  The gram-staining identification was observed to the strains isolated from all treatment plants. The gramstaining test such as gram-positive bacillus (GPB), gram-positive cocci (GPC), gram-negative bacillus (GNB) and gram-negative cocci (GNC) was determined in the bacterial strains that isolated both treatment plants. Figure 3 showed the gram-staining identification among the isolates from treatment plant. The total isolates (46 strains) were found to be 22 strains of GPB and GPC, 19 strains of GNP and GNC, 1 of both and 4 strains were undetermined. No result was found as the GNC in IWK treatment plant. The results indicated that the bioconversion of wastewater sludge might be effective through gram-positive and/or gram-negative strain especially isolated from IWK treatment plant as well as the IIUM treatment plants.

DISCUSSION
The microbial population have been measured in two treatment plants which were the average concentration of 1.7x10 3 cfu m 3 of mesophilic and 2.1x10 2 cfu m 3 of TSA-SB bacteria (bacteria associated with certain water born virulence factors) in the aeration tank of the activated sludge treatment plants and in the fixed film reactor 3x10 3 cfu m 3 of mesophilic and 730 cfu m 3 of TSA-SB bacteria were found [9] . The present study showed the higher density of bacterial community in the treatment plants compared to the literature.
Different applications of bacterial community isolated from wastewater sludge are employed globally. Seven groups of 26 morphological types of filamentous bacteria were isolated, cultivated and identified from activated sludge to achieve a better understanding of the complex phenomena of sludge bulking and foaming that influences the settleability and dewaterability of sludge [10] . A total of 165 denitrifying bacteria were isolated from activated sludge for polyphosphate accumulation and denitrification in biological removal of nutrient [11] . Several authors have been studied on biodiversity of bacterial community isolated from activated sludge [12][13][14] . So far no study was done on bioconversion of wastewater sludge with the potential bacterial culture in terms of effective biodegradability, dewaterability and settleability. Recently, filamentous fungi were isolated mainly from wastewater treatment plants for sludge bioconversion in liquid state (treatment) [15,16] and solid-state (compost) respectively [17] .
The results presented in this study indicated that 46 strains were isolated and purified from different sources in four treatment plants. The identification of gramstaining level was conducted in the isolates from IWK treatment plant. The higher microbial population was observed in the secondary clarifier in IWK treatment plant compared to other treatment plants. The potential bacterial strains through screening might enhance biodegradability and dewaterability of wastewater sludge considering its faster growth, resistance to contaminate and adaptation that could contribute the new development of biological treatment processes in future solutions.