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Article Released Mon-2nd-November-2009 07:16 GMT
Contact: Megawati Omar Institution: Universiti Teknologi MARA (UiTM)
 Microbes: sludge cleaners

Malaysian researchers found three strains of bacteria in petroleum sludge that could be used to quicken the rate of biodegradation .

Reported by Megawati Omar, Research Management Institute, UiTM, Malaysia

Petroleum refineries generate huge volumes of hazardous toxic petroleum sludge but now it seems that there is a possibility of cleaning this toxin organically. Malaysian researchers, student Noor Fazreen Dzulkapli with her three professors, Suhaimi Abdul Talib, Kalavathy Ramasamy and Yin Chun-Yang, of the Faculty of Civil Engineering, the Faculty of Pharmacy and the Faculty of Chemical Engineering of University of Teknologi MARA found that three strains of bacteria in petroleum sludge that could be used to quicken the rate of biodegradation in the slush.

It appears that microbes thrive in petroleum waste. The researchers saw a tapestry of microscopic wonders in this black mire, an activity of three microbial strains, Clavibacter michiganesis insidiosus, Rhodococcus rhodochrus and Brevibacterium otitidis which is able to remove hydrocarbon. Thus Noor Fazreen and her professors hypothesized that if microbes can flourish in the sludge, they could be useful to clean the sludge if the microbes are enhanced.

In their experiment, the microbes were taken from the sludge of a petroleum refinery in Terengganu, Malaysia. The microbes then were enriched in a standard culture enrichment technique in 21 days and the turbidity of the mediums was studied. The growth of microbes was monitored by comparing the medium’s turbidity in the control universal bottle against universal bottles with samples. The medium in the control universal bottle showed clear solution while the one in universal bottles with sample turned turbid. The turbidity of a medium indicates that enrichment is successful.

Next, the isolated the bacterial strains, 0.1 ml of culture from each universal bottle, was aseptically suspended in a 1-ml centrifuge tube containing 0.9 ml sterile ultra pure water. The mixture was homogenized via a vortex mixer where the serial dilution was made ranging 10-1 to 10-6. Then, each dilution was streaked on an agar plate, which was supplemented with hydrocarbon and later incubated for three weeks at 28 °C. After three weeks the dilution grew colonies of clear zone on the crystalline hydrocarbon layer. They were picked and cultured in a new Bushnell Hass medium for seven days then streaked on new Bushnell Hass agar. Only rapid growing, distinct, and separate morphologically unique colonies were selected for purification by repeated plating.

Clear yellowish, brownish or cream-colored zones appeared in the hydrocarbon-coated agar plates; showing formation of bacterial colonies and hydrocarbon degradation. Serial dilution in the range of 10-1 to 10-6 was prepared for each colony to decrease the intensity of microbes in each streaking. Decreasing the intensity of microbes in streaking, the better the purification process will be. At this point, fifty-three purified strains were obtained and labeled. Figure 1 illustrates the sample of the obtained colony.

Figure 1: Sample of obtained colony

Next was the hydrocarbon degradation test to see if these strains could remove hydrocarbon. In this test, eight hydrocarbon sources were used: n-decane, tetradecane, n-pentadecane, do-decane, anthracene, phenanthrene, dibenzothiophene and motor oil. The degradation results showed that a single species of microbes was able to remove several types of hydrocarbon. In this case, 8 strains of bacterial were able to remove hydrocabon from n-decane, 15 strains from tetradecane, 15 strains heptadecane, 13 strains on do-decane, 12 strains on anthracene, 12 strains on phenathrane and 17 strains on dibenzothiophene.

In this test, the strains that degraded hydrocarbon compounds were marked positive. Table 1 shows total positive scores of each hydrocarbon, showing hydrocarbon easiest to remove was heptadecane, followed by dibenzothiophene, phenanthrane, do-decane, anthracene, tetradecane and n-decane.

Table 1: Positive scores of each hydrocarbon

Hydrocarbon Positive scores
Heptadecane 61
Dibenzothiophene 59
Phenanthrane 57
Do-decane 51
Anthracene 49
Tetradecane 44
n-decane 31

Then the strains of microbe that can degrade polycylic aromatic hydrocarbon (PAH), especially anthracene, phenathrane and dibenzothiophene, were identified. PAH is a type of hydrocarbon with mutagenic and carcinogenic properties found most in petrochemical oil sludge.

The Biolog System Kit showed three strains that could degrade PAHs consistently. The three were Clavibacter michiganesis ss insidiosus, Brevibacterium otitidis and Rhodococcus rhodochrus.

Following, the study sees that the identification of these strains of bacteria is a breakthrough for a biodegradation process of the sludge. Cleaning the sludge organically means to clean the sludge by a biodegradation process - a breakdown of complex organic materials to forms carbon that can be used by other organisms. However, at root there is always a question of getting the correct agent to biodegrade the dirt. Here, it looks like researchers are making headway in finding cleaner microbes.

Reported by Megawati Omar, Research Management Institute, UiTM, Malaysia

Contact for further information
Faculty of Civil Engineering,
UiTM, Shah Alam, Malaysia

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