MOLECULAR CHARACTERIZATION OF A GREEN ALGAE ISOLATE BY 16S rRNA IN IMPROVEMENT OF CAROTENOID PRODUCTION

Sintesis karotenoid alami belum pernah melebihi produk sintetik pada skala komersial. Kurangnya pemahaman mengenai aspek mikrobiologis dan ekofisiologis isolat penghasil karotenoid seringkali menyebabkan terjadinya kesalahan penamaan spesies. Sam isolat lokal alga hijau dan Perairan Jepara yang digunakan sebagai pakan alami sumber karotenoid hewan-hewan perikanan, pada mulanya dianggap sebagai Dunaliella. Penamaan Dunaliella hanya dilakukan berdasarkan pengamatan mikrobiologis dan ekofisiologis yang kurang lengkap. Tujuan utama penelitian mi adalah menentukan spesies sam isolat lokal alga hijau secara molekuler menggunakan 16S rDNA untuk mendeteksi jalur biosintesis karotenoid yang digunakan. Urutan basa 16SrRNA yang diperoleh dianalisis menggunakan Multiple Alignment Analysis dan analisis filogenetik melalui program ClustalX ClustaiW, GeneDoc, Phylip dan NjPlot. Hasil penelitian memperlihatkan bahwa Isolat alga hijau menunjukkan similaritas yang tinggi dengan anggota-anggota Sianobakteria. Keserupaan tertinggi dimiliki dengan Cyanobacterium sp. MBIC 1021 sebesar 99 %, diikuti Synechoystis PCC6308 sebesar 95 %. Hasil analisis similaritas dan filogenetik memperlihatkan peluang bahwa Isolat alga hijau mengikuti jalur baru non-mevalonat dalam biosintesis karotenoidnya. Abstract Carotenoids production levels are not yet competitive with carotenoid levels presently produced by fermentation, synthesis and isolation. An attempt to optimize carotenoid production from local isolate of green algae from BBAP Jepara has faced several problems, primarily related to the microbiological and eco-physiological characteristic which affecting growth that have not sufficiently been understood. A misnamed of species also have arisen due to wrong characterization. One local isolate of Dunaliella species from BBAP Jepara was found potentially useful as source of carotenoids in food additives or as food supplement in fish farming. The present study aimed to characterize the species of green algae isolate from Jepara waters based on molecular techniques using 16S rRNA approach to detect its carotenoid biosynthetic pathway. Similarities analysis and phylogenetic relationship of 16S rRNA sequence was analyzing with Multiple Alignment Analysis by ClustalX ClustalW, GeneDoc, Phylip and NjPlot Programs. Molecular analysis showed close relationship among isolate of green algae and Cyanobacteria with 99 % similarity with Cyanobacterium sp. MBIC 1021 and 95 % similarity with Synechocysti.s PCC3O8. The result of this analysis indicated possibilities that a green algae isolate following the new non-mevalonate pathway for its catotenoid biosynthetics. Keywords A green algae isolate, Dunaliella, 16S rRNA, Cyanobacteri

Comparison of methods for genomic DNA isolation of rhizospheric microorganisms

A comparative study for the isolation of genomic DNA of rhizospheric microorganisms is described. The methods tested were based upon the methods of: (1) standard glass rod, (2) boiling glass rod, (3) Promega\u27s Wizard genomic DNA isolation method, and (4) agarose gel plug, with some modifications of each method. The efficiency and suitability of the method for genomic DNA isolation were analysed based on three parameters : (1) the level of DNA purity, (2) the concentration of DNA, and (3) the intactness of the DNA. It was observed that the standard glass-rod method gave the best results in terms of DNA purity and the DNA concentration obtained. However, in terms of DNA intactness, agarose gel plug method gave the best result compared to other methods. Keywords: genomic DNA - rhizospheric microorganism

Transformasi Gen Sucrose Phosphate Synthase {SoSPSl) Tebu (Saccharum officinarum L.) Untuk Meningkatkan Sevtesis Sukrosapadadaun Tembakau {Nicotiana tabacum L.)

INTISARI Sucrose phosphate synthase (SPS, EC 2.4.1.14) merupakan enzim utama yang berperan dalam biosintesis sukrosa pada tanaman dan menjadi elemen pokok interaksi antara fotosintesis dengan pertumbuhan. Tanaman tembakau {Nicotiana tabacum L.) ditransfonnasi dengan cDNA SPS tebu {Saccharum officinarum L.) yang diekspresikan dibawah kontrol promoter 35S CaMV. Tujuan penelitian ini adalah untuk mengetahui aktivitas SPS kandungan sukrosa daun tembakau transgenik yang ditransformasi dengan cDNA SPS tebu. Potongan daun tembakau diinfeksi dengan Agrobacterium: pKYS, diregenerasikan menjadi tanaman utuh {plantlet) dan diseleksi menggunakan media yang mengandung Kanamisin. Berdasarkan hasil analisis PCR dan western blot didapat 3 galur tembakau hasil transformasi yang mengandung gen (cDNA) SPS tebu, yaitu galur 3,13danl7. Dari ketiga galur tembakau transgenik, galur 13 mempunyai aktivitas SPS dan kandungan sukrosa daun tertinggi. Aktivitas SPS dan kandungan sukrosa daun pada galur 3 meningkat sebesar 65,76% dan 74,69% dari wild type. Hasil ini menunjukan bahwa cDNA SPS tebu yang dikonstruk pada pKYS dapat diekspresikan pada daun tembakau dan menghasilkan enzim SPS aktif secara biologi. Kata Kunci :Transformasi, Agrobacterium, pBI121, SPS, CaMV, Sukros

Cloning of betain aldehyde dehydrogenase gene of Escherichia coli and its use as a probe for related gene in salt-tolerant rhizospheric microorganisms

Betain aldehyde dehydrogenase gene (betB) of Escherichia coli has been PCR-amplified and cloned into pUC19 plasmid. The amplified fragment was subsequently labeled with digoxigenin and used as a probe for hybridization with either the genomic DNA of E. coli as well as with the rhizospheric microorganisms genome. The labeled probe has successfully detected the betB gene in the E. coli genome. The same probe, however, gave no positive signal when used for detection of related gene in the genome of several osmotolerant rhizospheric microorganisms isolates. Keywords: Betain aldehyde dehydrogenase â glycine betaine â rhizospheric microorganism

THE POTENCY OF COPPER-RESISTANT BACTERIA Cupriavidus sp. IrC4 ISOLATED FROM INDUSTRIAL WASTEWATER TREATMENT PLANT IN RUNGKUT-SURABAYA AS A BIOREMEDIATION AGENT FOR HEAVY METALS

Cupriavidus sp. IrC4 is a copper-resistant bacteria isolated from activated sludge in an Industrial Wastewater Treatment Plant in Rungkut-Surabaya, Indonesia. The purpose of this research was to study the potency of Cupriavidus sp. IrC4 as a bioremediation agent for copper, lead, mercury, and cadmium. Resistance of Cupriavidus sp. IrC4 to heavy metals were determined by measuring the minimum inhibitory concentration (MIC). Accumulation of copper, cadmium, and lead were determined by Atomic Absorption Spectrophotometer. Cupriavidus sp. IrC4 showed multiple resistance to heavy metals. The MICs of Cupriavidus sp. IrC4 to copper, lead, mercury, and cadmium were 16 mM, 15 mM, 6 mM, and 5 mM, respectively. The growth of Cupriavidus sp. IrC4 was inhibited by the addition of CuSO4 in the medium. The bacteria survived in the presence of high copper concentration as shown by the extension of the lag phase up to 36 hours. The analysis demonstrated that the copper resistance of the bacteria was facilitated through the accumulation of copper. Cupriavidus sp. IrC4 accumulated up to 367.78 and 260.01 mg/gram dry weight of cells of copper and lead, respectively. The bacteria demonstrated growth in the medium containing the mixture of 0.5 mM copper, lead, cadmium and accumulated those heavy metals up to 0.14, 24.74, and 12.49 mg/g dry weight of cells, respectively. The high resistance and capability of Cupriavidus sp. IrC4 to accumulate heavy metals can be exploited in bioremediation process for removing heavy metals from industrial sewage.Keywords: Accumulation, copper, Cupriavidus sp. IrC4,heavy metals, resistance

Characterization and Identification of Strain Km221, a Novel Mcpa Herbicide-degrading Bacterium Isolated From Coral Surface, Menjangan Kecil Island, Karimunjawa

In this study, bacterial strain KM221 was isolated from coral tissue in Menjangan Kecil Island, Karimunjawa, Indonesia. This strain is facultative anaerobic with MCPA (2-methyl-4-chlorophenoxy acetic acid) serving as the only known energy sources. Microscopy of isolate revealed that strain KM221 is gram-positive, catalase-positive, rod, spore-forming bacterium, motile, opaque, hair-like outgrowth and unpigmented colonies. The bacterium could not be identified on the basis of its carbon-source-utilization pattern, but a partial sequencing of the 16S rDNA analysis suggest that this strain is closely related to Bacillus iodinum.The ability to degrade MCPA herbicide was examined qualitatively in EMBA indicator medium. This bacterium grew exponentially with MCPA as the sole source of energy and carbon. The maximum growth rate (m max) and the saturated concentration on MCPA (Cs) were determined to be 0.8024 h-1 and 5.10 mg/l MCPA, respectively

Distribusi dan diversitas genetik bakteri diazotrof endofit pada tanaman tebu(Saccharum officinarum L)=Distribution and genetic diversity of diazotrhropic Endophytes in ...

ARDRA (Amplified rDNA Restriction Analysis) has been widely used to provide a relatively quick and simple analysis of microbial community diversity and phylogenetic relationship. A study has been carried out to determine the distribution and genetic diversity of diazotrophic endophytes in sugarcane root, stem, and leaves tissues using the ARDRA methodology based upon PCR (Polymerase Chain Reaction) amplification and RFLP (Restriction Fragments Length Polymorphism) analysis. Bacterial endophytes were isolated from sugarcane root, stem, and leaves tissues, followed by screening of diazotrophic bacteria using ARA (Acetylene Reduction Assay) analysis. The 16S rDNA was then PCR-amplified from the genomes of diazotrophic bacteria, followed by restriction enzyme analysis to create dendograms calculated based upon Dice Coefficient and UPGMA algorithm. The result of analysis demonstrated that eleven isolates of diazotrophic bacterial endophytes were distributed troughout root, stem, and leaves of sugarcane. The use of three different restriction enzymes, i.e. HaeIII, Hinfl, and RsaI was found to be sufficient in distinguishing the diversity among the isolates of diazotrophic bacterial endophytes. The dendogram produced based upon restriction enzyme analysis demonstrated no similarity between diazotrophic bacterial endophytes and thereference strains, i.e. either Azorhizophilus paspali DSM 2283T or ⢠Azotobacter chroococcum DSM 2286T. Similarity index produced was in the range of 0-100%. The highest similarity index (100%) was found among isolates ra9 and rbb9. It was assumed that the two isolates were the same and were found distributed in root and stem of sugarcane. Similarly, isolates ra12 and rdb12 also had 100% similarity index and were distributed in root and leaves of sugarcane. Keywords : diazotrophic endophytes, Saccharum officinarum, ARDRA, RFL

Molecular Detection of Endophytic Bacteria on Plantlet Tissue of Sugarcane

Endophytic bacteria live in plant host tissues without causing any symptoms. The aim of this study was to examine the indigenous endophytic bacteria on sugarcane plantlets produced from the young leaf cells by using tissue culture techniques. To detect the existence of endophytic bacteria in the plantlet tissue, a series of molecular method based on PCR were applied by using ribosomal intergenic spacer (RIS) primer followed by 16S rDNA partial sequence and single strand conformation polymorphism (SCCP). The results showed that the molecular method could detect the existence of bacteria in the tissues. Using the same methods, the bacteria were also found in other developmental stages of sugarcane (explants, differentiated tissues and callus). Key words: endophytic bacteria, PCR, RIS, 16S rDNA, SSC

Isolasi dan karakterisasi bakteri resisten tembaga dari limbah industri

ABSTRACT: Penelitian ini bertujuan untuk mengisolasi bakteri resisten tembaga dari limbah industri, menyeleksinya untuk memperoleh beberapa bakteri yang paling resisten, melakukan uji biokimiawi dan pengamatan morfologi koloni pada beberapa isolat terpilih. Hasil isolasi dan seleksi diperoleh 3 isolat bakteri yang paling resisten, dengan nilai MIC = 6-7 mM CuSO,. Bakteri tersebut merupakan bakteri gram negatif, dan dinamakan strain Cl, strain C2, dan strain C4. Penambahan 5 mM CuSO4 ke dalam medium menyebabkan perubahan morfologi koloni. Warna koloni berubah menjadi hijau dan struktur tepi koloni menjadi bergelombang. Perubahan morfologi ini diduga merupakan suatu mekanisme pertahanan diri yang dilakukan bakteri dalam menanggapi toksisitas tembaga. Kata kunci: Bakteri resisten tembaga, morfologi koloni, CuSO

Peningkatan Populasi Bakteri Pendegradasi Hidrokarbon Lumpur Minyak Oleh Penambahan Serasah Tanaman Penutup Tanah

ABSTRACT: A laboratory study was conducted with the aim of evaluating the effect of poultry manure and green manure additions on population growth of microorganisms involve in biodegradation of hydrocarbons contained in drill cuttings. The drill cuttinings was obtained from the Tanjung Santan, East Kalilmantan oil field of UNOCAL. The population growth was assayed by most probable number method on basal medium using extracted hydrocarbon as its sole carbon sources. Addition of green manure (Crotalaria juncea) enhanced population growth of hydrocarbon utilizing bacteria, while addition of poultry waste was not beneficial. Penelitian pada skala laboratorium telah dilakukan untuk mengetahui pengaruh pemberian kotoran ternak unggas dan pupuk hijau terhadap perubahan populasi mikroorganisme yang berperan dalam biodegradasi lumpur minyak. Lumpur minyak diperoleh dari lapangan minyak UNOCAI di Tanjung Santan, Kalimantan Tim ur. Perubahan populasi dikuti dengan cars "most probable number" dalam medium basal yang menggunakan hidrokarbon terekstrak sebagai satu-satunya sumber karbon. Penambahan pupuk hijau (Crotalaria juncea) memacu perkembangan populasi bakteri pengguna hidrokarbon sedangkan penambahan kotoran ternak unggas tidak memberikan pengaruh yang menguntungkan. biodegradai hidrokarbon yang teradpat dalam lumpur minyak. Lum bahan organik terhadap perubahan populasi mikroorganisme yang berperan dalam biodegradasi hidrokarbon yang terdapat dalam lumpur minyak. Penambahan pupuk hijau (Crotalaria jurtcea) memacu perkembangan populasi bakteri pengguna hidrokarbon, sedangkan penambahan kotoran ternak unggas tidak memberikan pengaruh yang menguntungkan