Isolation and identification of strain
B. salmalaya was originally isolated from indigenous agricultural soil in Malaysia (2.99917°N, 101.70778°E). New strains were isolated with Brain–Heart–Infusion (BHI) agar as a highly nutritious growth medium which contains sodium chloride 5 g, disodium phosphate 2.5 g, dextrose 2 g, brain heart (infusion solids form) 8 g, agar 13.5 g, pancreatic digest of animal tissue 5 g, pancreatic digest of Casein 16 g, and supplemented with 5% sheep blood in 1 l. Among the isolated strains, 139SI exhibited strong hemolytic activity. On the basis of 16S rRNA sequencing and phylogenetic analysis, this strain was designated as a novel strain. Further information and molecular analysis of strain 139SI were reported recently [18, 19].
Leachate sample
Leachate samples were obtained from an untreated leachate pond at the Bukit Beruntung landfill in Malaysia (3°25′32.14″N, 101°32′56.6″E); this landfill is a non-sanitary, open dumping and an operational landfill. Bukit Beruntung landfill (operated in 1992), which is visible from the North-South Highway, receives approximately 80 t of waste daily. The depression in the hilly area allows the expansion of the disposal site. Lack of enforcement by local municipality resulted in illegal dumping of municipal solid waste along the road to the landfill site.
Determination of the minimum inhibitory concentration of NH3–N
To determine whether B. salmalaya strain 139SI is resistant to ammonia nitrogen, the following test was performed. First, strain pellets were prepared by centrifuging culture broth (OD600 = 1.0) at 3500 rpm for 15 min. These pellets were added into 10 mL of leachate with 1000 ppm concentration of NH3-N and incubated at 35 °C and 150 rpm for 72 h. Subsequently, bacteria were harvested and resuspended in leachate, which contained 2000 ppm ammonia nitrogen. The bacterial/leachate mixture was incubated under the above conditions. This process was repeated at different concentration and it was completed when the final concentration of NH3-N in the landfill leachate was 100 g/l. Approximately 20 μL of the each above mixture was spread onto a plate of solid medium and incubated at 35 °C for 16 h. The culture medium comprised BHI and leachate in 2:3 ratio and was solidified with agar. Pure colonies were selected, cultured, and stored for further experimental work.
Bioaugmentation process
Pure B. salmalaya strain 139SI colony was cultured in BHI culture medium for 24 h at 33 °C and 150 rpm. Afterward, the biodegradation experiment was performed with 30 mL of landfill leachate inoculated with 3 mL bacterial strain (OD600 = 1) and incubated at 30 °C for 11 days at 150 rpm. Samples were collected and analyzed at 1, 3, 5, 8, and 11 days. To test the biodegradation capability of B. salmalaya strain 139SI and to remove the effect of indigenous bacteria, sterilized landfill leachate was degraded under same conditions.
Effects of environmental conditions on bioaugmentation
To determine the effect of various conditions on the degradation efficiency of B. salmalaya strain 139SI, experiments were carried out at different temperatures (30 °C, 35 °C, and 40 °C), pH values (6, 7, and 8), and inoculum dosage (1, 3, and 5 mL). Experiments were performed in triplicate. Design-Expert Software was used to analyze experimental data. Significant effect on biodegradation was determined at 95% level (P < 0.05) using SPSS version 18.
Elemental analysis
The decreases in the values of NH3–N, COD, and BOD were measured to evaluate the effectiveness of the microbial treatment of landfill leachate. COD concentration was determined on the basis of the agreed methods for Water and Wastewater examination (APHA 5220C). Samples were diluted and added to standard COD ampules and incubated in a dry incubator at 150 °C for 2 h. The COD tubes were allowed to cool down to room temperature and titrated with 0.05 M ferrous ammonium sulfate. NH3–N concentration was measured by APHA 4500 NH3 F-Phenate method. The sample was transferred to a 50-mL conical flask and mixed with the following solutions: phenol (1 mL), sodium nitroprusside (1 mL), and oxidizing solution (2.5 mL). The sample was covered and allowed to settle for at least 1 h and to develop color at room temperature (22–27 °C) in subdued light. Subsequently, the absorbance was read at 640 nm using spectroquant PHARO 100 [4]. BOD concentration was measured using APHA 5210 B method. Removal percentage was computed on the basis of the concentration measurement using the following equations:
$$ {\mathrm{Removal}}_{\mathrm{COD}}\left(\%\right)=\left[\left({\mathrm{COD}}_{\mathrm{i}}\hbox{--} {\mathrm{COD}}_{\mathrm{f}}\right)/{\mathrm{COD}}_{\mathrm{i}}\right]\times 100\%, $$
(1)
$$ {\mathrm{Removal}}_{\mathrm{N}\mathrm{H}3\hbox{--} \mathrm{N}}\left(\%\right)=\left[\left({\mathrm{N}\mathrm{H}}_3-{\mathrm{N}}_{\mathrm{i}}\hbox{--} {\mathrm{N}\mathrm{H}}_3-{\mathrm{N}}_{\mathrm{f}}\right)/{\mathrm{N}\mathrm{H}}_3-{\mathrm{N}}_{\mathrm{i}}\right]\times 100\%. $$
(2)
where i and f are the initial and final concentrations, respectively.
The morphology of cell surfaces (before and after adsorption) was evaluated with scanning electron microscopy (SEM, SEI quanta SEG 450, Netherlands).
