Beberapa Gen pada Bakteri yang Bertanggung Jawab Terhadap Produksi Bioetanol

Harga minyak mentah dunia yang berfluktuasi dan cadangan minyak yang makin menipis telah mendorongpenggunaan biofuel sebagai bahan bakar alternatif dan mengintensifkan penelitian bioetanol dengan menggunakanmikroba. Identifikasi dan karakterisasi gen-gen yang bertanggung jawab dalam produksi bioetanol (etanologen) danekspresinya pada beberapa inang telah dilakukan untuk meningkatkan produksi bioetanol oleh mikroba. Tulisan inimengulas sumber-sumber gen bioetanol (pdc dan adh) dan ekspresinya pada beberapa bakteri inang sebagai genyang berdiri sendiri maupun sebagai operon dalam memproduksi bioetanol. Piruvat dekarboksilase (pdc) danalkohol dehidrogenase (adh) adalah gen-gen yang bertanggung jawab dalam produksi bioetanol yang ditemukanpada bakteri mesofilik Zymomonas mobilis dan ragi Saccharomyces cerevisiae. Sumber gen pdc lebih terbatasdibandingkan dengan gen adh. Gen pdc belum ditemukan dalam jaringan hewan maupun bakteri termofilik.Manipulasi genetik beberapa bakteri dengan pdc dan adh yang diisolasi dari beberapa bakteri telah dilakukan untukmeningkatkan produksi bioetanol dari sumber karbon yang murah, yaitu biomassa. Ekspresi etanologen telahberhasil dilakukan pada bakteri mesofilik, baik gram-positif maupun gram-negatif. Namun, ekspresi etanologenpada bakteri termofilik perlu penelitian lebih lanjut untuk mencapai keberhasilan yang tinggi

INHIBITION OF THE GROWTH OF TOLERANT YEAST Saccharomyces cerevisiae STRAIN I136 BY A MIXTURE OF SYNTHETIC INHIBITORS

Biomass from lignocellulosic wastes is a potential source for biobased products.  However, one of the constraints in utilization of biomass hydrolysate is the presence of inhibitors. Therefore, the use of inhibitor-tolerant microorganisms in the fermentation is required. The study aimed to investigate the effect of a mixture of inhibitors on the growth of Saccharomyces cerevisiae strain I136 grown in medium containing synthetic inhibitors (acetic acid, formic acid, furfural, 5-hydroxymethyl furfural/5-HMF, and levulinic acid) in four different concentrations with a mixture of carbon sources, glucose  (50 g.l-1) and xylose (50 g.l-1) at 30oC. The parameters related to growth and fermentation products were observed. Results showed that the strain was able to grow in media containing natural inhibitors (BSL medium) with µmax of 0.020/h. Higher level of synthetic inhibitors prolonged the lag phase, decreased the cell biomass and ethanol production, and specific growth rate. The strain could detoxify furfural and 5-HMF and produced the highest ethanol (Y(p/s) of 0.32 g.g-1) when grown in BSL. Glucose was utilized as its level decreased in a result of increase in cell biomass, in contrast to xylose which was not consumed. The highest cell biomass was produced in YNB with Y (x/s) value of 0.25 g.g-1. The strain produced acetic acid as a dominant side product and could convert furfural into a less toxic compound, hydroxyl furfural. This robust tolerant strain provides basic information on resistance mechanism and would be useful for bio-based cell factory using lignocellulosic materials.

Pertumbuhan Padi Varietas Ciherang Setelah Diinokulasi dengan Azospirillum Mutan Multifungsi

Modern agriculture is very closely related to the application of fertilizer to induce plants grow. The application of bio-fertilizers is expected to reduce the negative impact of chemical fertilizers. The purpose of this study was to determine the effect of multi-functional Azospirillum N2 fixation, P solubility and IAA production on the growth of Ciherang rice in pot experiment in greenhouse BB Biogen. The experiment treatment were 3 types of inoculation (non-inoculation, inoculation using wildtype Aj Bandung 6.4.1.2 and the mutant isolate of AJM 3.7.1.14 ), and 4 levels of fertilizer application (non-fertilization, a quarter dose, a half dose, and the real dose of fertilization on rice in lowland). The Azospirillum isolates were used wildtype isolate Aj Bandung 6.4.1.2 and mutant isolate AJM 3.7.1.14 that was isolated and mutated using ethyl methanesulfonate (EMS) in BB Biogen. Seeds of Ciherang rice were inoculated using Azospirillum at cell density 106 cell/ml in different seedling tray. After 14 days, the seedlings were transferred to planting pots which consist of 3 plants per pot. Parameters observed were plant height, number of tillers, number of panicles per hill, wet and dry weight of panicles per hill, weight of 100 seeds, N and P content of the stover. The results showed that both wild-Azospirillum and mutant inoculum had no effect on the vegetative growth of Ciherang, but showed significant effect on the number of panicle per hill, grain weight per hill and dry weight of seeds per panicle. The use of Azospirillum and N fertilizer combination affected the growth and rice yields, also reduced chemical fertilizer application

KINETIC EVALUATION OF ETHANOL-TOLERANT THERMOPHILE Geobacillus thermoglucosidasius M10EXG FOR ETHANOL PRODUCTION

Thermophiles are challenging to be studied for ethanol production using agricultural waste containing lignocellulosic materials rich in hexose and pentose. These bacteria have many advantages such as utilizing a wide range of substrates, including pentose (C5) and hexose (C6). In ethanol production, it is important to use ethanol tolerant strain capable in converting lignocellulosic hydrolysate. This study was aimed to investigate the growth profile of ethanol-tolerant thermophile Geobacillus thermoglucosidasius M10EXG using a defined growth medium consisted of single carbon glucose (TGTV), xylose (TXTV), and a mixture of glucose and xylose (TGXTV), together with the effect of yeast extract additionto the media. The experiments were conducted at the School of Biotechnology and Biomolecular Sciences of The University of New South Wales, Australia on a shake flask fermentation at 60°C in duplicate experiment. Cultures were sampled every two hours and analised for their kinetic parameters including the maximum specific growth rate (µmax), biomass yield (Yx/s), ethanol and by-product yields (acetate and L-lactate) (Yp/s), and the doubling time (Td). Results showed that this strain was capable of growing on minimal medium containing glucose or xylose as a single carbon source. This strain utilized glucose and xylose simultaneously (co-fermentation), although there was glucose repression of xylose at relatively low glucose concentration (0.5% w/v), particularly when yeast extract (0.2% w/v) was added to the medium. The highest biomass yield was obtained at 0.5 g l-1 on glucose medium; the yield increased when yeast extract was added (at 0.59 g l-1). The highest specific growth rate of 0.25 was obtained in the phase I growth when the strain was grown on a mixture of glucose and xylose (0.5% : 0.5% w/v) medium. Diauxic growth was shown on the mixture of glucose, xylose, and yeast extract. The strain produced low level of ethanol (0.1 g l-1), as well as low level (0.2 g l-1) of by-products (L-lactate and acetate) after 15 hours. The results suggests its potential application for fermenting lignocellulosic agricultural wastes for ethanol production

Construction and Expression of Pet Operon using Shuttle Vector for Mesophilic and Thermophilic Bacteria

Keuntungan fermentasi etanol pada suhu tinggi mendorong penelitian perakitan bakteri termofilik etalogenik. Selain itu, kemampuan bakteri termofilik dalam penggunaan gula pentosa hasil degradasi biomasa memberi peluang untuk menekan biaya produksi bioetanol. Tujuan dari penelitian ini adalah untuk mengkonstruksi pet (production of ethanol) operon dengan menggunakan shuttle vector pMK18 dan melihat ekspresinya dalam bakteri mesofilik dan termofilik. Konstruksi dan ekspresi pet operon dengan menggunakan adhT dari bakteri termofilik dan pdc dari bakteri mesofilik, dan penggunaan mesofilik-termofilik shuttle vector sebagai backbone-nya baru pertama kali dilaporkan. Pet operon adalah suatu susunan gen penyandi produksi etanol yang terdiri dari gen pdc (pyruvate decarboxylase) dan adh (alcohol dehydrogenase). Konstruksi pet operon menggunakan gen adhT dari bakteri termofilik Geobacillus thermoglucosidasius M10EXG dan pdc (pyruvate dehydrogenase) dari bakteri mesofilik Zymomonas mobilis ZM4 telah dilakukan dengan menggunakan mesofilik-termofilik shuttle vector pMK18. Ekspresi pet operon pada bakteri mesofilik Eschericia coli dapat memproduksi 0,3 g/l etanol dengan aktivitas adhT sekitar 0,02 U/mg protein dan aktivitas pdc sekitar 0,004 U/mg protein. Perlu dilakukan penelitian lanjutan untuk perbaikan konstruksi pet operon untuk sistemtermofik pada Thermus thermophilus HB27, karena konstruksi yang didapat belum optimum untuk sistem termofilik ini. Hasil ini diharapkan akan mengawali pengembanganteknik manipulasi genetik pada bakteri termofilik yang masih sangat terbatas, khususnya pengembangan teknik manipulasi termofilik etanologenik. Kata kunci: Etanol, bakteri termofilik, bakteri mesofilik, pet operon, ekspresi gen.

MULTIFUNCTIONAL MUTANTS OF Azospirillum sp. WITH ENHANCED CAPABILITY OF SOLUBILIZING PHOSPHORUS, FIXING NITROGEN AND PRODUCING INDOLE ACETIC ACID

Azospirillum sp. have long been known as biofertilizer for plant growth because of its capability to produce phytohormones and fix nitrogen from the atmosphere. Multifunctional Azospirillum strain Aj Bandung 6.4.1.2 isolated in 2009 from cauliflower (Brassica oleracea) rhizosphere in Lembang, Bandung, West Java, was capable of fixing nitrogen, solubilizing tricalcium-phosphate, and producing phytohormone indole acetic acid (IAA). The study aimed to modify the multifunctions of Azos-pirillum sp. for better capability of fixing N2, solubilizing P, and producing IAA using ethyl methanesulfonate and 1-methyl-3-nitro-1-nitrosoguanidine (EMS) mutagen. The study was conducted at Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD) in 2010. The results showed that this strain was genetically mutagenized using EMS for better performance in solubilizing P, fixing N2 (nitrogenase activity), and producing phytohormone (IAA). The optimum concentration and the length of incubation time for the process have been determined. Nine selected mutants with increasing capability to solubilize P (determined by clear-zone formation on Pikovskaya’s medium) have been characterized for nitrogenase activities and IAA production compared to wild type Aj Bandung 6.4.1.2. The effect of mutagenesis on IAA produc-tion and nitrogenase activities varied among the mutans. Two mutants, AzM 3.7.1.16 and AzM 1.7.2.12, showed superiority in the production of IAA, while two mutants, AzM 1.5.1.14 and AzM 3.7.1.15, were superior in nitrogenase activities. The EMS mutagenesis of Azospirillum sp. showed enhanced dissolving capa-bility of unsoluble phosphate (tricalciumphosphate) and increased IAA production and nitrogenase activity.

GROWTH IMPROVEMENT OF TOMATO WITH THE APPLICATION OF BACTERIAL ISOLATES PRODUCING INDOLE ACETIC ACID (IAA) AND PHOSPHATE SOLUBILIZER

Soil bacteria have important roles in biogeochemical cycle for soil fertility and have been manipulated for ecologically-friendly crop production.  The search for beneficial association between microbes and plants for promoting growth and health should be studied for tomato growth improvement. The study aimed to  evaluate 19 microbial isolates which produced indole acetic acid (IAA) affecting growth and development of tomato (Palupi variety), and  molecularly identify the most effective isolates in improving tomato growth based on 16s rDNA sequences. The experiment was conducted in pots using a complete randomized design with three replications. The parameters observed included plant height, plant dry weight, root length, root dry weight, and fruit fresh weight.  The isolates that significantly improved tomato growth were molecularly identified using 16s rRNA sequence. The phenotypic properties such as IAA content and phosphate solubilizing index (PI) of the superior isolates were determined. Results showed that the application of bacterial isolates on tomato significantly increased plant dry weight and fruit yield. From 19 isolates tested, Aj 3.7.1.14 significantly increased plant dry weight, root length, and fruit yield. This isolate produced IAA of about 14.77 ppm and PI of 1.86.  Molecular analysis on Aj 3.7.1.14 demonstrated that the isolate had 89% similarity to Pseudomonas fragi. The identified P. fragi was found to be the most effective isolate for improving tomato growth and fruit yield. Another isolate, Bacillus amyloliquefaciens was found to promote root length, root dry weight, and fruit yield. These isolates are potential to be further investigated for field trial

Penentuan Alergenisitas Protein Gen RB pada Kentang Produk Rekayasa Genetika Berdasarkan Studi Bioinformatika

Genetically modified products (GMP) of Katahdin potato event SP951 containing RB gene resistant to late blight diseasescaused by Phytophtora infestans has been developed in the USA. This Katahdin SP951 potato has been crossed with localvarieties Atlantic and Granola for its development in Indonesia. In the release process, the GMP potato should be tested forenvironmental and food safety. One of the food safety assessment needs to be done by determining allergenicity of RB proteinwhether it is potential as allergen. This research aims to translate the RB gene sequence into RB protein sequence andinvestigate the potential RB protein as an allergen through bioinformatic studies. This study was performed based on thealignment with available protein allergens from available database websites. The predicted RB protein obtained from 2,913amino acids RB gene was a 971 amino acids length protein with ATG as a start codon and TAA as a stop codon. Bioinformaticsstudies of RB protein were performed using www.allergenonline.com, consisted of three searches, i.e. full-length search byFASTA, 80 amino acids search by FASTA, and 8 amino acid exact matches. For full-length alignment search, there are threeallergen proteins similar with RB protein sequence with the percentage identity of <35%, while for alignment with 80 aminoacids and 8 amino acids did not show similarity with any allergen protein in the database. It can be concluded that RB proteindid not have any potential as an allergen, as according to Codex Alimentarius guidelines for full-length alignment search, onlyprotein with identity greater than >50% indicating possible cross reactivity with protein allergen

GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations

PENINGKATAN PERTUMBUHAN PADI VAR CIHERANG SETELAH DIINOKULASI DENGAN Azospirillum MUTAN MULTIFUNGSI PENAMBAT N2, PELARUT P DAN PENGHASIL FITOHORMON INDOLE ACETIC ACID (IAA).

Modern agriculture rellies on the application of fertilizer to enhanched plant growth. Excessive application of chemical fertilizers in the long term will decrease soil fertility, therefore the use of biological fertilizers is expected to reduce the negative impact. The aim of this study was to determine the effect of multi-functional Azospirillum for N2-fixation, phosphate solubilizer  and producing phytohormone Indole Acetic Acid (IAA) on the growth of rice plants Ciherang in the pot experiment conducted in the BB Biogen’s greenhouse. Two treatments were tested: types of inoculation (not inoculated, were inoculated with the wildtype AjB 6.4.1.2 and inoculated with mutant isolates AJM 3.7.1.14), and 4 levels of fertilizer application (not fertilized, a quarter dose of paddy fertilizer recommendations, a half dose of fertilizer rice in the rice fields, and the appropriate dose of fertilizer in paddy fields). Azospirillum used is wildtype isolate AjB 6.4.1.2 and mutant isolate AJM 3.7.1.14 isolated and and mutated in BB Biogen. Ciherang rice seed inoculated with Azospirillum on the cell density of 106 cells / ml at the different seedlings tub. After the age of 14 days after planting, the seedlings were transferred into pots with three plants per pot. Parameters measured were: plant height, number of tillers, number of panicles per clump, fresh weight and dry panicles per clump, weight of 100 grains, and the content of N and P stover. Results show that inoculantion, affect significantly on the number of panicles per hill, grain weight per panicle and the dry weight of grains per panicle.