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Mokhzanni Mustapa1, Nor Jannah Sallehudin1, Mohd Shamzi Mohamed2, Normawaty Mohammad Noor3 and
Raha Ahmad Raus1
1Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia, Kuala Lumpur,
Malaysia
2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, UPM
ABSTRACT
Direct sampling of microalgae from nature inevitably brings together the problem of massive growth of bacteria
and fungi. In this study, bacterial and fungal contamination level was evaluated from the Chlorella sp. isolated from two
different locations of local freshwater area. Attempts to obtain axenic Chlorella sp. culture by combinations of antibiotic
and antifungal at different range of concentration treatment were investigated. It is evident that there were three different
bacteria and two different fungi present in the culture, but apparently sterility can be achieved when ampicillin, cefotaxime
and carbendazim cocktail were employed at concentrations of 700 µg/mL, 200 µg/mL and 0.1µg/mL, respectively. These
concentrations are also proven harmless toward Chlorella sp. as higher concentrations inhibit the growth of microalgae. It
was found that by streaking the contaminated microalgae twice onto the TAP agar containing the previously described
cocktail, completely removed the contaminating fungi and bacteria from the culture. In conclusion, this study suggested
that axenic Chlorella sp. can be attained with this method and cocktail recipe.
Keywords: microalgae, Chlorella sp., bacterial contamination, fungal contamination, ampicillin, cefotaxime, carbendazim, cocktail.
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inhibited the growth of Chlorella sp. When tested at a Initial screening to obtain bacterial-free culture by
lower concentration, 0.1 µg/mL carbendazim showed antibiotic combination was conducted with broad
inhibition of white fungal growth but only partial spectrum antibiotics of ampicillin and cefotaxime at a
inhibition of the black fungal growth. While, at the same concentration range of 100-1000 µg/mL and 50-200
concentration, TPM and benomyl completely inhibited the µg/mL, respectively. The inhibitions of both antibiotics
growth of both types of fungus. Interestingly, at this are dose dependent since reduced and no bacterial colonies
concentration, carbendazim allowed vigorous growth of were observed as the concentration increased (Table 3).
the microalgae, unlike TPM and benomyl. This finding is Based on Table 3, ampicillin at 700 µg/mL and cefotaxime
in agreement with previous studies which demonstrated at 200 µg/mL were chosen to be added for the
that the sensitivity and response of antifungal toward decontamination cocktail.
microalgae vary by species. For instance, 1 mg/L of each Ampicillin is broad-spectrum penicillin, which is
carbendazim, thiram, tebucunazole have been proven safe part of aminopenicillin under the β-lactams group.
for Chlorella sp. whereas carbendazim, thiophanate Ampicillin is known to be effective against both gram-
methyl, and benomyl were harmless for Chlamydomonas negative and gram-positive organisms. It provides
reinhardtii up to 160mg/L [6] [9]. Although carbendazim bactericidal effect via inhibition of bacterial cell wall
at 0.1 µg/mL did not provide strong fungicidal effect, it synthesis by binding to one or more of the bacterial
allows Chlorella sp. to grow unlike the other two penicillin binding proteins (PBPs). Since the cell walls in
antifungal. Based on these, carbendazim at 0.1 µg/mL is the growing bacteria are constantly being synthesized,
used to make up the cocktail for decontamination of inhibition of synthesis is effective at controlling growth.
Chlorella sp. from fungus. Ampicillin is known to be effective towards Gram-
A closer observation on the black fungal growth positive: Streptococcus spp., Enterococcus spp., Listeria
on the TAP agar plate containing 0.1 µg/mL carbendazim monocytogenes, and Gram-negative: H. influenzae, E. coli,
showed that the black fungus demonstrated arrested Proteus mirabilis, Salmonella spp., Shigella spp. [11].
filament structures on the agar surface whereas at Cefotaxime is a third generation cephalosporin, a
concentrations of 1 µg/mL and above, no fungal growth group of β-lactams antibiotic and exerts bactericidal
was visibly detected. These observations indicated that activity, like penicillin. However, cefotaxime expresses
various processes such as intracellular transport, resistancy to β-lactamase enzymes which are produced by
maintenance of cell shape and cellular mobility through bacteria to inhibit the bactericidal effect [12]. Due to these
cilia and flagellar action were disrupted when 14C of reasons, the combination of ampicillin and cefotaxime is
carbendazim completely binds to the microtubule of the expected to bring a synergistic effect to the cocktail
fungus for complete growth inhibition [10]. treatment.
(-)Not Detected, (+) Limited growth (++) Moderate growth (+++) Abundant growth
(NC) Not Conducted (-) Not Detected, (+) Limited growth (++) Moderate growth (+++) Abundant growth
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(-)Not Detected, (+) Limited growth (++) Moderate growth (+++) Abundant growth
700/200/0.1 700/200/0.1
Control Plain agar
(first treatment) (second treatment)
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700/200/0.1 700/200/0.1
Control Plain agar
(first treatment) (second treatment)
A clean Chlorella culture was obtained after two Kulliyah of Engineering, International Islamic University
serial cocktail treatments with ampicillin, cefotaxime and Malaysia, Gombak.
carbendazim at concentrations of 700 µg/mL, 200 µg/mL
and 0.1 µg/mL. However, before the second cocktail REFERENCES
treatment was carried out, single colonies from the first
cocktail treatment plate had to be cultured individually in
[1] Guo Z. and Tong Y.W. 2014. The interactions
TAP broth to ensure clean microalgae. Restreaking on the
final plain agar after the second cocktail treatment is between chlorella vulgaris and algal symbiotic
essential to ensure no impending bacteria or fungi were bacteria under photoautotrophic and
formed before mass inoculation. Similar procedure for photoheterotrophic conditions. Journal of Applied
serial cocktail succession prior to restreaking the cells onto Phycology. 26:1483-1492.
plain agar were reported in the decontamination study of
marine microalga, Tetraselmis sp. and green alga [2] Wasanasathian A. and Peng C.A. 2007. Algal
Chlamydomonas reinhardtii [8] [13]. There is also photobioreactor for production of lutein and
evidence that after the decontamination, the colour of the zeaxanthin. Bioprocessing for Value-Added Products
colonies differed between untreated and treated, where the
from Renewable Resources. pp. 491-505.
latter was a darker green. The fact that there is variable
reaction between microalgae species, higher concentration
of carbendazim usage in the cocktail at above 10 µg/mL [3] He P.J., Mao B., Lu F., Shao L.M., Lee D.J. and
has a high potential to be an alternative isolation technique Chang J.S. 2013. The combined effect of bacteria and
in a cross-contaminated culture especially between chlorella vulgaris on the treatment of municipal
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568.
CONCLUSIONS
The cocktail combination of ampicillin [4] Dube J.F. 1952. Observations on a chlorophyll-
(700 µg/mL), cefotaxime (200 µg/mL) and carbendazim deficient strain of chlorella vulgaris obtained after
(0.1 µg/mL) has proven to be an appropriate cocktail treatment with streptomycin. Science. 116: 278-279.
solution to decontaminate infected Chlorella sp. culture
without affecting the growth. It is possible this similar [5] Ma J., Zheng R., Xu L. and Wang S. 2002.
method can be utilized for other microalgae species as Differential sensitivity of two green algae,
well.
scenedesmus obliqnus and chlorella pyrenoidosa, to
12 pesticides. Ecotoxicology and Environmental
ACKNOWLEDGEMENTS
Safety. 52(1):57-61.
This research is supported by RACE 14-005-
0011 under the Ministry of Higher Education, Malaysia
and by the Biotechnology Engineering Department,
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