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A review on efficacy of genus Pseudomonas in hydrocarbons degradation

N. H. SHAHIDAN

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia

Abstract

Pseudomonas is well-known as an effective hydrocarbon degrader. This potential of Pseudomonas species has led to several strains being genetically engineered to assist in cleaning up contaminated site. The efficacy of Pseudomonas mechanism in hydrocarbon biodegradation is largely depending on its ability to form biosurfactant called rhamnolipid. Surface active molecule rhamnolipid acts as a booster in increasing the rate of hydrocarbon consumption. However, environmental factors such as pH, temperature and salinity have significant effects on rhamnolipid action. Since Pseudomonas can also produce extracellular enzymes such as lipase, its potential as a hydrocarbon degrader surpasses other bacterial genera such as Micrococcus, Aeromonas and Serratia.

1) Introduction

Being ubiquitously found in soil and marine environment, genus Pseudomonas has been classified as one of the most effective oil degraders (Hong et al, 2005), along with other bacterial genera such as Micrococcus, Bacillus, Aeromonas, Serratia and Proteus (Benka-Coker & Ekundayo, 1996; Rahman et al, 2003). The most widely studied Pseudomonas aeruginosa has a good reputation as the most powerful oil degrader among Pseudomonas species. Tang et al (2006) reported P. aeruginosa has the capability to consume on long-chain length alkenes and able to survive for at least five years at oil contaminated site, without facing detrimental effects on its morphology and size. As universal oil degrader, some strains of Pseudomonas have been used commercially to alleviate contaminated sites that have hydrocarbons as main pollutants through bioremediation technologies (Evans et al, 2004). Hydrocarbon products contain an intricate mixture of different components, both aromatic and aliphatic hydrocarbons. Kerosene for instance, consist components ranging from C8 to C18 n-alkenes, as well as aromatic groups. In contrast, gasoline has aromatic groups such as toluene, dimethylbenzene and trimethylbenzene as major components, whilst having alicyclic hydrocarbons in small amount (Wongsa et al, 2004). A study conducted by Wongsa et al (2004) once again discovered P. aeruginosa was the best kerosene degrader. On the other hand, based on study done by Barathi and Vasudevan (2001), P. fluorescens was shown to effectively utilize hexadecane, hexane, decane, kerosene and crude oil.

Extraordinary mechanisms of biodegradation carried out by Pseudomonas lead to its effectiveness in utilizing hydrocarbon as energy source. This review will highlight the efficacy of Pseudomonas by exploring its degradation mechanisms in general context; particularly on the production of rhamnolipid and secretion of extra cellular enzymes, mainly lipase.

2) The production of rhamnolipid by Pseudomonas

Rhamnolipid, a biosurfactant synthesized by some Pseudomonas strains has been proven to be essential in assisting the degradation of hydrocarbons, especially alkenes (Tang et al, 2006). By definition, biosurfactant is a “group of surface active molecules” that is naturally produced by microorganisms to enhance biodegradability of hydrocarbon-based-substrates (Tang et al, 2006; Illori et al, 2005). Barathi and Vasudevan (2001) mentioned that biosurfactant is not just potentially limited to breaking down aliphatic hydrocarbons such as alkenes, but also recalcitrant polyaromatic compounds such as naphthalene and phenanthrene.

Inakollu et al (2004) stated that a number of strains of Pseudomonas characteristically capable of producing biosurfactants. Other than P. aeruginosa, P. fluorescens can also form biosurfactant before the degradation of oil occurs (Evans et al, 2004; Barathi & Vasudevan, 2001). The availability of biosurfactant increases the surface area-in-contact for bacterial enzymatic activities (Wongsa et al, 2004). Inakollu et al (2004) added by minimizing the surface tension and droplet size, biosurfactants stimulate the degradation in natural way. Other organisms such as Torulopsis bombicola and Mycobacterium spp. also form biosurfactant (Ilori et al, 2005). The degradation of crude oil, pyrene and phenanthiene for instance are speed up effectively due to the presence of rhamnolipids manufactured by P. aeruginosa (Tang et al, 2006; Inakollu et al, 2004; Cybulski et al, 2003). Tang et al (2006) also mentioned that the buildup of rhamnolipids might have a major impact on the enhancement of crude oil degradation; this is strongly supported by relative correlation between emulsification time and initial amount of rhamnolipids secreted by P. aeruginosa ZJU.

3) Factors affecting the effectiveness of rhamnolipid actions

3.1 Gene regulation

The mechanism of gene regulation enables production of rhamnolipid to be controlled. Ochsner et al (1994) stated that rhlR gene is essential in synthesizing rhamnolipids since the interruption at this locus had contributed to the formation of “rhamnolipid-deficient mutant strains”. Tang et al (2006) also agreed the production of rhamnolipid is controlled by gene expression, since there is no significant relationship between amount of biomass generated and rhamnolipids production. rhlR gene that controls the productivity of rhamnolipids might be highly expressed due to environmental stress impact on quorum sensing (Tang et al, 2006).

3.2 Structure of rhamnolipid

The structure of rhamnolipid is one of the possible factors contributing to the successful hydrocarbon degradation by Pseudomonas. Different bacterial species and unique structure of an emulsifier are two variables affecting the efficacy of hydrocarbon utilization (Cybulski et al, 2003). Cybulski et al (2003) highlighted there is a strong correlation between the structure of emulsifier and rate of hydrocarbons biodegradation, even among Pseudomonas strains. P. aeruginosa strain, for example is shown to conduct emulsification better than P. putida.

Figure 1. Chemical structures of rhamnolipid (Adapted from Urum et al., 2006)

The significance of rhamnolipid structure in assisting the degradation is not fully understood. However, it can be concluded that the structure of surfactant does affect the magnitude of hydrocarbon degradation as different surfactant with different structure have different effectiveness level (Cybulski et al, 2003).

3.3 Environmental factors

Environmental factors play a crucial role in influencing the productivity and efficacy of rhamnolipids. In general, as a biosurfactant, rhamnolipid activity is controlled by environmental conditions such as pH, salinity and temperature (Ilori et al, 2005; Inakollu et al, 2004; Jirasripongpun, 2002). Ilori et al (2005) pointed out that the chemical structure of biosurfactant gives an apparent benefit for hydrocarbon degradation and very unlikely to be disrupted due to extreme temperature and pH. This applies to rhamnolipid as well (Figure 1). In addition, as biosurfactant is synthesized naturally by microorganisms, the production of toxic is minimized. Benka-Coker and Ekundayo (1996) highlighted the amount of oil might affect the biodegradation rate as well, corresponding to poor aeration due to the lack of oxygen. Plus, while the action of Pseudomonas in degrading oil is accelerated through the formation of rhamnolipid, size and structure of hydrocarbon substrates may conversely slow down the breaking down processes. However, the ability of Pseudomonas to secrete lipase enzyme will overcome this difficulty.

4) Comparison of the effectiveness in rhamnolipid action to other biosurfactants

Noordman and Janssen (2002) claimed the degradation of hydrocarbon, hexadecane by rhamnolipid has higher rate compared to other biosurfactants. Direct contact between bacterial cells and hydrocarbon droplets is almost certainly due to the minimization of hydrophobicity of cell surface “after the extraction of lipopolisaccharide from the cellular envelope by rhamnolipid”, hence accelerate the microbial degradation of that particular hydrocarbon. Urum et al (2006) on the other hand conducted a study comparing the effectiveness of biosurfactant rhamnolipid, saponin and sodium dodecyl sulfate (SDS). He found out rhamnolipid and saponin are almost equal in degrading crude oil, whilst SDS as the most inefficient. Nevertheless, in this case rhamnolipid is still the best biosurfactant since it is produced naturally via microbial activity, while saponin need to be extracted from quillaja bark (i.e from plant source) and SDS is a synthetic surfactant.

5) Secretion of extracellular enzymes

Pseudomonas has the capability of manufacturing few types of extracellular enzymes, and one of them is lipase. Extra cellular lipase of Pseudomonas is characteristically unique amongst other lipases produced by other microorganisms, especially its ability to resist high temperature and enzymatic action in basic environment. Therefore, Pseudomonas lipase is very suitable to be used in bioremediation application to degrade esters and lipid wastes (Hasanuzzaman et al, 2004). Vasileva-Tonkova and Galabova (2002) pointed out the process of breaking down organic pollutants mainly carried out by microbial community via the secretion of hydrolytic enzymes. Hence, along with the formation of rhamnolipid, Pseudomonas has an apparent advantage compared to other bacterial genera as a hydrocarbon degrader since it can both secrete enzymes and feed on hydrocarbons. Vasileva-Tonkova and Galabova (2002) stated that not all bacterial genera have the ability to grow on hydrocarbon as energy source, and at the same time secrete hydrolytic enzyme. This kind of microorganism like Pseudomonas appears to be rare in environment. Catalytic action mechanism of Pseudomonas is varied even amongst species, contributing to dynamic cooperation amongst Pseudomonas species themselves during the process of degradation. Lipase of P. mendocina strain 3121-1 was found to be different from lipase of other P. mendocina which is capable of acting on wide range of substrates (Bendikiene et al, 2004). Since the secretion of lipase is likely to be suppressed by sugars and other intricate substrates, hydrocarbon might be more suitable for lipase break down activity (Kanwar & Goswami, 2002).

Conclusion

Two main mechanisms in hydrocarbon degradation conducted by Pseudomonas give this genus a unique ability to degrade hydrocarbon based pollutant either naturally in environment or for commercial purposes. In other words, the use of Pseudomonas either wild-type or genetically modified strains in bioremediation technologies to clean up contaminated sites is just perfect and suit the nature best.

REFERENCE:

Barathi S. and Vasudevan N. (2001). Utilization of petroleum hydrocarbons by Pseudomonas fluorescens isolated from a petroleum contaminated soil. Environment International 26: 413-416.

Benka-Coker M. O. and Ekundayo J.A (1996). Applicability of evaluating the ability of microbes isolated from an oil spill site to degrade oil. Environment Monitoring and Assessment 45: 259-272.

Bendikiene V., Surinenaite B., Juodka B., and Safarikova M.. (2004). Insights into catalytic action mechanism of Pseudomonas mendocina 3121-1 lipase. Enzyme and Microbial Technology 34: 572-577.

Cybulski Z., Dziurla E., Kaczorek E., and Olszanowski A. (2003). The influence of emulsifiers on hydrocarbon biodegradation by Pseudomonadacea and Bacillacea Strains. Spill Science and Technology Bulletin 8: 503-507.

Evans F.F, Seldin L., Sebastian G.V, Kjelleberg S., Holmstrom C. and Rosado A.S. (2004). Influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16S rRNA based-PCR and DGGE. Letters in Applied Microbiology 38: 93-98

Hasanuzzaman M., Umadhay-Biones K. M, Zsiros S.M., Morita N., Nodasaka Y., Yumoto I., and Okuyama H. (2004). Isolation, identification and characterization of a novel, oil-degrading bacterium, Pseudomonas aeruginosa T1. Current Microbiology 49: 108-114.

Hong J.H., Kim J.,Choi O.K.,Cho K.S. and Ryu H.W. (2005). Characterization of a diesel-degrading bacterium, Pseudomonas aeruginosa IU5, isolated from oil-contaminated soil in Korea. World Journal of Microbiology and Biotechnology 21: 381-384.

Ilori M.O, Amobi C.J. and Odocha A.C. (2005). Factors affecting biosurfactant production by oil degradating Aeromonas spp. isolated from a tropical environment. Chemosphere 61: 985-992.

Inakollu S., Hung H.C., and Shreve G.S (2004). Biosurfactant enhancement of microbial degradation of various structural classes of hydrocarbon in mixed water systems. Environmental Engineering Science 21: 463-469

Jirasripongpun K. (2002). The characterization of oil-degrading microorganisms from lubricating oil contaminated (scale) soil. Letters in Applied Microbiology 35: 296-300.

Kanwar R. and Goswami P. (2002). Isolation of a Pseudomonas lipase produced in pure hydrocarbon substrate and its application in the synthesis of isoamyl acetate using membrane-immobilised lipase. Enzyme and Microbial Technology 31: 727-735.

Noordman W.H and Janssen D.B (2002).Rhamnolipids stimulates the uptake of hydrophobica compounds by Pseudomonas aeruginosa. Applied and Environmental Microbiology 68: 4502-4508.

Ochsner U.A., Koch A.K., Fiechter A. and Reiser J. (1994). Isolation and characterization of a regulatory gene affecting rhamnolipid surfactant synthesis in Pseudomonas aeruginosa. Journal of Bacteriology 176: 2044-2054.

Rahman K.S.M., Rahman T.J., Lakshmanaperumalsamy P., Marchant R. and Banat I.M. (2003). The potential of bacterial isolates for emulsification with a range of hydrocarbon. Acta Biotechnology 23: 335-345.

Tang X., Zhu Y. and Meng Q. (2006). Enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU after preservation in crude oil-containing medium. World J Microbiol Biotechnol 22: 1-8.

Urum K., Grigson S., Pekdemir T., and McMenamy S. (2006). A comparison of the efficiency of different surfactants for removal of crude oil from contaminated soils. Chemosphere 62:1403-1410.

Vasileva-Tonkova E. and Galabova D. (2002). Hydrolytic enzymes and surfactants of bacterial isolates from lubricant contaminated wastewater. Z Naturforsch 58: 87-92.

Wongsa P., Tanaka M., Ucno A., Hasanuzzaman M., Yumoto I. and Okuyama H. (2004). Isolation and characterization of novel starins of Pseudomonas aeruginosa and Serratia marcescens possessing high effiency to degrade gasoline, kerosene, diesel oil and lubricating oil. Current Microbiology 49: 415-422.

nak isi borang SM2008 atau SM2007?

Nabi saw menegaskan :

ما من مَوْلُودٍ إلا يُولَدُ على الْفِطْرَةِ فَأَبَوَاهُ يُهَوِّدَانِهِ أو ينصرانه أو يمجسانه
Tiadalah seorang anak yang dilahirkan melainkan ia dilahirkan di atas fitrah . Maka ibubapanya bertanggungjawab menjadikan anaknya Yahudi , Nasrani atau Majusi .
( Riwayat Imam Bukhari dan Muslim )

Semalam saya berchatting dengan sahabat lama saya yang sudah lama tidak bertemu secara online. Dia menyindir-yindir saya. Nasihatilah sahabatnya, gunakan bahasa-bahasamu yang indah. Dia sedia memberitakan saya email sahabatnya itu, saya kata, bukan saya yang sepatutnya menegur orang itu, tetapi dialah yang sepatutnya. Dia menukar topik lain pula – dia ini memang suka mengelak apabila saya ingin berbicara tentang hal sebegini.

      Saya tahu dia resah. Fail exam, terpaksa pula extend sampai setahun lagi. Dia accident dengan kereta mewah, dia menanggung hutang lagi. Terpaksa kerja untuk bayar hutang, duit hilang pula. Sekarang dah masuk musim sejuk, dia hilang kerja. Kena cari kerja lain. Hidupnya bagai sudah jatuh ditimpa tangga, terkena batu pula kepala. Bertimpa-timpa. Dia kesal dengan hidupnya. Dia kata badluck. Saya bilang, Allah telah menyediakan semua ini sebagai tarbiyyah dariNya supaya dia lebih tabah lagi masa depan. Saya bilang, cuba check balik, solat, mengaji Al-Quran, puasa sunat, adakah kita jaga semua itu? Dia kata, semua orang tabligh sama saja, suka cakap benda yang sama. Saya bilang, saya bukan tabligh, tapi ini adalah realiti. Dia kata, orang kuat ibadat pun kena problem juga. Benar, kata saya, tapi cara mereka handle problem itu yang berbeza. Anda semua setuju dengan saya? Cubalah hayati ayat di bawah ini:-

“Tidak ada sesuatu kesusahan (atau bala bencana) yang ditimpakan di bumi dan tidak juga yang menimpa diri kamu, melainkan telah sedia ada di dalam Kitab (pengetahuan Kami) sebelum Kami menjadikannya; sesungguhnya mengadakan yang demikian itu adalah mudah bagi Allah. (Kamu diberitahu tentang itu) supaya kamu tidak bersedih hati akan apa yang telah luput daripada kamu dan tidak pula bergembira (secara sombong dan bangga) dengan apa yang diberikan kepada kamu dan (ingatlah), Allah tidak suka kepada tiap-tiap orang yang sombong takbur, lagi membanggakan diri” (Al-Hadid:22-23)

Sahabat saya memberitahu saya dahulu dia budak baik. Maksud dia, dia sukakan aktiviti bercorak Islam, bukan macam sekarang, rasa ingin melarikan diri. Dia tidak suka mereka yang suka memaksa-maksa. Saya bilang, setiap orang approachnya berbeza-beza, ubahlah pemikiran kita yang mereka cuma ingin mendekatkan kita kepada Islam.  Setiap harakah berbeza cara kerjanya. Benar, attitude kita lebih terasa kesannya daripada kata-kata. Lafaz yang dizahirkan melalui amalan itu lebih berkesan. Ini bukan satu kesangsian. Ini adalah kenyataan.

    Memang banyak yang menjadi bahan bualan kami. Masalah Melayu-Cina, asal usul Melayu, Parameswara, keunggulan Melayu satu masa dahulu, rumput dan sebagainya. Dia kata buat ape nak tarik orang Islam ke Islam, tarik orang kafir ke Islamlah. Konsepnya begini, orang Islam kita ajak ke aktiviti-aktiviti Islam supaya bertambah kefahamannya terhadap Islam, itu priority kita sekali. Umat Islam sekarang hancur, sahsiah hancur, itu semua kita sudah sedia maklum. Jadi fiqh auliyatnya sudah lain, kita tarbiyyah diri dan orang Islam dahulu, baru kita pergi kepada orang kafir. Kalau orang Islam sendiri tidak boleh dijadikan contoh, bagaimana orang kafir ingin tertarik kepada Islam? Seperti yang saya katakana tadi, perbuatan itu lebih kuat kesannya dari kata-kata. Ada faktor-faktor lain yang menjadi punca Islam adalah agama yang paling cepat berkembang waktu ini. Tapi ini bukan menjadi topik tulisan saya hari ini.

       Orang Islam sekarang masih mencari-cari identiti diri, sedangkan Islam terbentang di depan mata. Masih melarikan diri dari realiti. Masih tenggelam dengan penipuan duniawi yang serba indah penuh permainan yang warna warni. Namun semuanya punah apabila nyawa tercabut dari jasad. Antara mahu dengan tidak, itu sahaja. Tidak ada alasan tidak dapat belajar tentang Islam, bahan bersepah-sepah. Antara hati mahu atau tidak, itu saja.

        Dia kata dia tak suka mengaji, tapi suka dengar bacaan Al-Quran. Dia rasa dirinya rosak. Tapi bila tengok orang lain, dia rasa orang lain lebih teruk. Saya bilang, dalam hal harta dunia, kita kena tgk orang yg jauh lebih rendah dari kita. Untuk hal akhirat, kita kena tgk orang yg atas dari kita. Dia kata, Well said. Okay, dia sudah nampak point di situ. Dia bilang nasyid sekarang bukan lagi nasyid seperti dulu. Nasyid sekarang nasyid rap. Nasyid sekarang ini semuanya sampah, itu dia kata. Saya menyetujuinya dengan bersungguh-sungguh.

Akhirnya saya berjaya memperkenalkan dia dengan www.searchtruth.com. Dia kata ini website yang hebat, bagus sekali. Saya tersenyum puas. Saya doakan dia selalu menggunakan website ini kelak.

    Tiba-tiba dia bertanya saya adakah saya suka mendengar Al-Quran dan siapa recitor kegemaran saya. Saya jawab Mishary (http://alafasy.tripod.com/) Dia kata dia suka Saad Al-Ghamidi saja.  Saya offer dia mahukah dia mendengar Mishary pula, dia jawab ya. Saya gembira kerana dia juga suka recitor ini. Senang katanya, bacaan jelas tidak bersambung-sambung. Dia gembira benar, dia ingin seluruh Al-Quran itu. Alhamdulillah. Dia kata, natsukashi, maksudnya satu perasaan yang sudah lama kita  tak dengar dan dengar, kita akan rasa perasaan ini. Satu perasaan yang puas, mungkin. Alhamdulillah. Perbualan kami terhenti apabila dia suruh saya berhenti. Dia kata saya ini bahaya. Sedangkan jin pun masuk Islam apabila dengar Al-Quran, apatah lagi manusia.

Marilah kita kembali ke fitrah kita – fitrah yang sentiasa inginkan kepada kebaikan.

Taqaballah huwa min kum

Saya memang suka membaca, tetapi saya tidak suka menghafaz. Bodoh benar saya ini. Allah menggegarkan hati saya tentang pentingnya membaca, menghafaz dan seterusnya menzahirkan ilmu saya dalam amalan melalui “The Alchemist of Al-Ghazali”. Tahukah anda bahawa Imam Al-Ghazali dahulu hanya suka menuntut ilmu tetapi tidak menghafaz ilmu-ilmunya? Hingga pada suatu ketika dia dan rombongan yang diikutinya dirompak ketika dalam kembara ilmunya ke Baghdad. Dia mengenggam erat kertas-kertas yang ditulis hasil pembelajarannya dengan gurunya. Kata-kata perompak itu memberinya satu kesedaran untuk satu anjakan paradigma. “Ilmu itu seharusnya berada di hati, bukannya di atas helaian kertas”. Teringat pula kata-kata Saidina Ali, “Janganlah melihat siapa yang berkata-kata tetapi lihatlah apa yang dikatakannya”.

Supp exam saya dijangka 12 Disember nanti insya-Allah. Ini kali pertama saya tidak ambil exam pada hari yang sepatutnya. Takut, risau, perasaan saya bercampur aduk. Perancangan Allah itu adalah yang terbaik. Moga Dia memudahkan segalanya.  Ampunkan saya pacal yang hina dina ini.

شَرَعَ لَكُم مِّنَ الدِّينِ مَا وَصَّى بِهِ نُوحًا وَالَّذِي أَوْحَيْنَا إِلَيْكَ وَمَا وَصَّيْنَا بِهِ إِبْرَاهِيمَ وَمُوسَى وَعِيسَى أَنْ أَقِيمُوا الدِّينَ وَلَا تَتَفَرَّقُوا فِيهِ كَبُرَ عَلَى الْمُشْرِكِينَ مَا تَدْعُوهُمْ إِلَيْهِ اللَّهُ يَجْتَبِي إِلَيْهِ مَن يَشَاءُ وَيَهْدِي إِلَيْهِ مَن يُنِيبُ

Allah telah menerangkan kepada kamu di antara perkara-perkara agama yang Ia tetapkan hukumnya apa yang telah diperintahkanNya kepada Nabi Nuh dan yang telah Kami (Allah) wahyukan kepadamu (wahai Muhammad) dan juga yang telah Kami perintahkan kepada Nabi Ibrahim dan Nabi Musa serta Nabi Isa, iaitu: Tegakkanlah pendirian agama dan janganlah kamu berpecah belah atau berselisihan pada dasarnya. Berat bagi orang-orang musyrik (untuk menerima agama tauhid) yang engkau seru mereka kepadanya. Allah memilih serta melorongkan sesiapa yang dikehendakiNya untuk menerima agama tauhid itu dan memberi hidayat petunjuk kepada agamaNya itu sesiapa yang rujuk kembali kepadaNya (dengan taat).

Malaysia gempar dengan BERSIH. Aku masih belum ada masa untuk update diri tentang ini. Banyak benar yang ingin ku tulis. Namun tiada masa. Exam sungguh hampir. Rindu sungguh pada ayah yang pandai berjenaka. Pada ibu yang cekal melayan kerenah kami adik-beradik. Pada ancik yang paling iras perangainya dengan aku. Pada Ein yang suka buat perangai, tapi masih adik kesayanganku. Pada adik bongsu Humairah yang suka mengomel. Pada rumah kami yang indah dengan pohon-pohon kelapa kuning renek. Tersergam. Pada desa yang sawah padinya terbentang luas. Tempat bermain kami waktu kecil. Namun Tok telah pergi meninggal kami. Moga Allah menempatkannya di kalangan orang-orang yang mendapat rahmatNya. Kalian, tunggu aku pulang!

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