**6.1 Enrichment and isolation of carbazole degrading bacteria**

Many different types of bacteria exist in our natural environment according to Zulkharnain and Taka [37]. Although most research focus on isolating heterocyclic hydrocarbon degrading bacteria from sewage or site with high probability of contamination, these degrading bacteria could be found anywhere in the environment and not just in certain area. Therefore, lake environment is chosen to discover

different type of novel carbazole degrading bacteria. The project started by taking lake water from Unimas west campus lake, Sarawak to isolate heterocyclic hydrocarbon mainly carbazole degrading bacteria. Sampling was done at the side of the lake closer to land as the number of bacteria are higher at coastal, shallow area [38]. Once, sample was taken, enrichment was done using minimal salt media (MSM) with carbazole as sole carbon source where the degradation of carbazole could be observed in **Table 6**. After around three to four weeks of the first enrichment, color change could be observed where the enrichment turned to clear yellow from cloudy white before. Here, based on study by Zulkharnain and Taka [37], it proves that the bacteria present in the enrichment had utilize the carbazole as sole carbon source and degrade it causing color change. It also explains that the accumulation and production of different metabolites during degradation caused the color change [37]. Another research by Stope et al. [39] and Maeda et al. [40] explained that the change in color of the enrichment was due to the meta cleavage product formation which are the intermediates. When second and third enrichment was done, color change could be observed in a faster rate within two weeks as degradation rate increases. According to the concept where 3-ring structure is more stable and recalcitrant than 2d-structure, bacteria takes about 10 days to degrade fully [37].

From the enrichment, MSM agar plates supplemented with carbazole were prepared and used in this experiment to provide a selective condition and avoid growth of any other bacteria [41]. Double layer agar was used in this experiment to observe the clear zones easily. Streaking was done to observe the growth and degradation on MSM agar plates. After two weeks, clear or hollow zones could be observed on the plates in **Figure 4**. According to Takahashi et al., [42], clear zones that appear on the bacterial colony is because of the mineralization of insoluble CAR by bacteria. As clear zone could be observed, this proves the ability or capability of bacteria to degrade the substrate and utilize it as sole energy source.

Once clear zone was observed, sub-culturing was done to isolate pure colony of bacteria that shows degradation where six bacteria were isolated and designated as IM1, IM2, IM3, IM4, IM5 and IM6 strains. Based on the result shown, all the bacteria were able to grow on MSM agar supplemented with carbazole plate. Although, not all the six isolated showed clear zones or halo formation, the isolates were able to grow on the plates proving the capability of using the substrate. To observe the production of intermediate product and confirm the degradation, the isolated bacteria were inoculated on MSM liquid media as in liquid media it is easier to degrade the substrate because the bacteria becomes free moving cell and it mimics the original or natural condition of the bacteria in lake water. The MSM culture with bacteria was placed on orbital shaker to make sure the bacteria get enough nutrient as the shaker moves in circular motion.

#### **6.2 Gram staining**

The isolated bacteria were then characterized morphologically by gram staining. Based on results in **Table 7**, all isolated bacteria (IM1, IM2, IM3, IM4, IM5, IM6) were identified as Gram negative, rod shaped bacteria after viewing under light microscope with 100X magnification with immersion oil. To differentiate between Gram-positive and Gram-negative bacteria, it has been known that gram staining is the best and easiest method so far. Bacteria that can retain the primary strain which is crystal violet (purple color) are Gram-positive bacteria while Gram-negative bacteria classifications are the ones that could not retain the primary strain, where it will stain pink with safranin instead [43]. Based on a few studies, some Gramnegative bacterium such as *Pseudomonas sp.* LD2 [44], *Achromobacter sp.* Strain CAR1389 [45] and *Sphingomonas sp.* GTIN1 1 [46] has been reportd to utilize and

degrade carbazole. This shows that most or many of the heterocyclic hydrocarbon degrading bacteria are Gram-negative.

#### **6.3 Molecular characterization**

The molecular characterization was done firstly by DNA extraction procedure based on method in 3.6.1. Detergent which is SDS and proteinase K was used to break the cells and digest contaminated protein in the cell. The digestion of protein process was done under optimal condition of 37°C and incubated where the tubes were flipped every 10 minutes to increase digestion. Once the mixture became viscous, it shows that the digestion process has ended. To take up the DNA in aqueous phase, phenol: chloroform: isoamyl (25:24:1) was added and placed on rotary shaker to maximize the bacterial DNA take up. Centrifugation was done to form the double layer of organic and aqueous phase and to separate the upper layer containing DNA into new tubes. Next, isopropanol was added before centrifuging leaving the precipitate of DNA at the side and bottom of the tube. For further amplification using PCR machine, the DNA precipitate was redissolved in TE buffer.

According to method in 3.6.2. amplification was done using thermal cycler before 16S rDNA sequencing to identify the bacteria. In the PCR amplification, three main process were involved which are denaturation, annealing and extension phase which is all repeated for 30 cycles, therefore producing a million copies in around 2 hours. As shown in AGE analysis in the results, all six isolates (IM1, IM2, IM3, IM4, IM5, IM6) was successfully amplified using 27F as forward and 1492R (reverse) primers showing clear, intact band which is located at 1500 bp after being compared with 1Kb DNA ladder in lane 1. According to Angeline [47], the total of two primers and the sequence of the target DNA is the size of the PCR products.

One of the most common technique to separate DNA was used in this experiment which is gel electrophoresis. In this experiment, according to method in 3.6.3., 1% of gel was used with 1 ml of 50X TAE and 50 ml of distilled water. As the resolution of DNA band depends on the concentration of gel, most concentration of the gels are made in range 0.7% to 2% [48]. The four major factors that influence the migration rate of DNA, according to Cheng and Zhang [48] are, the concentration of agarose where lower concentration will produce better results for large DNA fragments. Then, the size of DNA is also a major factor where smaller fragments tend to migrate in a faster rate compared to larger fragments. Furthermore, the DNA conformation where DNA in supercoiled form is faster than in linear form. Lastly, during electrophoresis, the voltage supplied plays important role as well as the lower the voltage the slower the migration rate. After the bands of PCR were viewed under UV light, the remaining products were purified using the right protocol before sending for sequencing to identify the isolated carbazole degrading bacteria.

#### **6.4 The identification of isolated bacteria**

The sequence data for strain IM1, IM2, IM3, IM4, IM5 and IM6 was obtained from the First BASE Laboratories Sdn Bhd which are shown in **Table 8**. All the sequence data were analyzed using MEGA software before the species were determined molecularly using BLAST programming from NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi).

From the analysis, it is shown that the signals for IM1 and IM3 signs were not strong enough resulting in lower identification percentage. For IM1strain, the BLAST result showed that the bacteria belong to the genus of *Bradyrhizobium sp.* with 90% identity compared to the database. *Bradyrhizobium sp.* is a gram negative, nitrogen fixing bacteria that is rod in shape. It is most commonly found in soil and it

is slow growing compared to *Rhizobium sp.* A study by Qu and Spain [49], showed the first clear evidence for the initial steps in biodegradation of nitroanilines by *Bradyrhizobium JS329.* Although not many studies could be found on degradation of carbazole by *Bradyrhizobium sp.,* a study on isolation and characterization of species that degrades polycyclic and heterocyclic aromatic compounds under extremely low oxygen conditions, found that Shinorhizobium sp. C9 (AF227756), Mesorhizobium amorphae (AJ271899), Mesorhizobium sp. SH2851 (AY141983) and Rhizobium ciceri (U07934) has the ability to degrade heterocyclic hydrocarbons [35].

For IM2 strain, it was discovered that the bacteria belong to the genus *Ochrobactrum sp.* with 99% identity to the database while IM6 was also discovered to be another type of *Ochrobactrum* species with a specified name which is *Ochrobactrum anthropi sp*. with 100% identity. This species is known to be gram negative, rod shaped, non-pigmented, aerobic bacteria. This explains how the two isolates could be from the same species as it is hard to differentiate when the bacteria are not pigmented. Novel strain of *Ochrobactrum anthropi* HM-1 has been isolated from oil-contaminated soil where the degradation potential has been reported that contributes to bioremediation of used engine oil polluted sites [50].

With similarity of 97% to the database, IM3 strain was found to be *Pseudomonas aeruginosa sp. this* species is a pathogenic bacterium that could cause disease to plant, animal and even human. This bacterium is also a gram negative, rod shaped bacteria that could be found widely in environment, especially in soil, water and plants. In an experiment where potent biodegradation of crude oil was assessed, *Pseudomonas aeruginosa sp. have* been reported to degrade at the percentage of 58% of crude oil with direct or indirect assistance of glycerol or rhamnolipid. As *Pseudomonas aeruginosa sp. has* the ability to utilize vegetable oil or glycerol as sole carbon source [51]. Crude oil is mainly involved in biodegradation as the major component of crude oil are volatile hydrocarbon that needs to undergo biodegradation.

IM4 and IM5 strain has been confirmed to belong to the genus of *Burkholderia sp.* with 100% similarity to the database after the 16S rDNA sequencing was done. *Burkholderia sp.* are obligate aerobic, gram-negative bacteria that is well known for their antibiotic resistance. Some family of *Burkholderia sp.* such as *Burkholderia mallei sp.* are known to be pathogenic to mostly horses and other related animals. Based on a study by Inoue [52], 27 carbazole utilizing bacteria was isolated from environment where three of them were *Burkholderia sp. strain* NE-7, *Burkholderia sp.a and Burkholderia sp. strain* NW-1. When hybridization was done, CAR gene was found in these isolates proving the ability to utilize carbazole as sole carbon source.

#### **6.5 Gas chromatography-flame ionization detector (GC-FID) analysis**

As the rate of degradation cannot be determined through direct observation, GC-FID analysis was used in order to obtain the result of the degradation rate of carbazole by the six (IM1, IM2, IM3, IM4, IM5, IM6) isolated bacteria. The GC-FID machine was used by analyzing the residual substrate after 12 days incubation period of the bacteria supplemented with carbazole where pattern of degradation was observed and compared. In previous study by Okoh et al. [53], the degradation of crude oil by Pseudomonas aeruginosa was determined by measuring the reduction rate of crude oil. Hedlund et al. [54] had also done analysis using GC-FID where the degradation rate of substrate was calculated by looking at the disappearance of substrate.

In this experiment, the sample were analyzed every two days until day 12. Based on the chromatogram observed from the analysis result in 4.4, the peak of substrate, CAR was detected at the retention time from 14.0 to 15.0 minutes and the pattern of CAR utilization are revealed to vary among all isolates. This could be due to the sundry nature or environment of hydrocarbon present at the location from which the isolates were taken [55]. For IM1 strain, the percentage of residual carbazole was seen to be decreasing from 10.87% on day 3 to 0% on day12 while the degradation percentage showed gradual increase from 89.13% on day 3, 95.14% on day 6, 97.71% on day 9 and 100% on day 12. This shows that *Bradyrhizobium sp.* possess the ability to degrade carbazole gradually with highest percentage on day 12 resulting on zero amount of carbazole left.

For IM2 strain, the percentage of residual of CAR was high on day 3 with 94.35% but reduced greatly to 16.13% on day 6 and subsequently to 12.7% on day 9 and 4.63% on day 12. For the degradation rate, on the third day, the degradation rate was low at 5.65% but increase sharply on day 6 up to 83.87 and 87.3% on day 9 followed by 95.37 on day 12. Therefore, the degradation rate was slower on the first 3 days and increased a lot from the sixth day. However, it could be observed that this *Ochrobactrum sp.* does not totally degrade CAR after 12 days as there was still some residual substrate left. Thus, indication could be made by observing the plotted degradation graph that the strain might need more time to completely degrade the substrate compared to other bacteria.

The percentage of residual substrate for IM3 isolate, decreased from 96.57% on day 3 followed by 60.51% on day 6 and 57.67% on day 9. Whereas the degradation percentage on day 3 was low at 3.43% and increased to 39.49% on day 6, 42.33% on day 9 and finally to 100% on day 12. The *Pseudomonas aeruginosa sp. which* had been widely analyzed as a degrader showed that the strain took 12 days to completely utilize and degrade carbazole as sole carbon source based on the analysis result.

For IM4 strain, it could be observed from the result that the degradation rate was one of the highest compared to other isolates. The percentage of residual carbazole reduced to 14.06% on day 3 and was fully utilize leaving 0% from day 6 onwards. The degradation rate went down to 85.94% on the third day and increase fully to 100% on day 6. For IM5 strain which was identified as the same species with IM4, the rate of degradation however was different from IM4 strain with residual substrate percentage of .38.46% on day 3 and gradually falling to 36.9% on day 6, 6.96% on day 9 and was totally utilized by day 12. The degradation rate on day 3 was 61.545 which was higher compared to IM4 strain and gradually decreased to 93.04% on day 9 and 100% on day 12. Thus, both IM4 and IM5 strain which was identified as *Burkholderia sp.* could have been different type of stains showing different level of degrading ability.

The IM6 strain which was identified as *Ochrobactrum anthropi sp.* has the highest degradation rate based on the GC-FID analysis result where it was shown that the degradation rate on day 3 was up to 91.22% and the substrate, CAR was totally degraded by day 6 onwards. The residual substrate left on day 3 was 8.78% from 100% on the first day and by day 6 there was no substrate left. Based on study by Abulgasem Alenabi [56], the degradability of bacteria on CAR or other substrate was believed to be influenced by the adaptability of isolates to utilize the substrate as sole carbon source. The optimum growth factors such as temperature, pH and presence of nutrients also plays an important role on the capability of bacteria to degrade substrate [57].

#### **7. Conclusion and recommendations**

As a conclusion, all the objectives were successfully achieved. The appearance of clear zone on MSM agar plates and color change in MSM broth of all six isolates proved to be carbazole degrading bacteria as they were able to utilize the substrate carbazole as sole carbon source. All six species of bacteria isolated from Unimas lake

water was successfully identified using 16S rDNA sequencing which was found to be *Bradhyrhizobium sp., Ochrobactrum sp., Burkholderia sp*. and *Pseudomonas aeruginosa sp. all* these bacteria were found to be gram negative bacteria through gram staining analysis. As for the second objective of measuring the degradation rate, all the six isolates were able to degrade carbazole after 12 days of incubation when tested using GC-FID analysis where *Burkholderia sp*. and *Ochrobactrum sp.* showed high degradation rate. Thus, this proves that all the six species are a good candidate for bioremediation.

For the recommendations, the bacterial growth parameter such as pH, agitation and temperature can be manipulated to increase the degradation rate on the substrate used. The degrading ability of isolated bacteria strains could also be tested with other heterocyclic compound such as dibenzofuran and dibenzothiophene. Next, when preparing and handling samples for GC-FID analysis, precautions should be applied in order to gain more accurate results. Moreover, in order to fully understand on the degradation pathway of isolates, experiment on detection of metabolites produced can be done in the future.
