Formation Of Clear Zone Research Articles

Amylase Zymography.

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Isolation and Screening of Potential Cellulase Free-Xylanase Producing Bacterial Strains from Cow Dung

Research on xylanase has markedly increased due to its potential applications in pulping and bleaching processes using cellulose free preparations, textile processes, enzymatic saccharification of lignocellulosic materials and waste treatment. The objective of present study was to isolate and screen potential microbes from cow dung with respect to cellulase free xylanase production. Twelve isolates were obtained from cow dung with different frequencies of occurrence. Among the different culture isolates, the selection of selective isolate was done depending upon the clear zone formation on xylan agar medium. Submerged fermentation was done with the selective isolates to identify the cellulase free xylanase stains at pH 7.0 and at temperature 37°C. Bacillus sp. X6 was capable of producing xylanase of 15.32 IU/ml using basal salt medium under submerged fermentation

Susceptibility of methacrylate-based root canal filling to degradation by bacteria found in endodontic infections

To present a case of endodontic failure obturated with a methacrylate-based root filling material, Resilon/RealSeal (RS). To determine if RS is susceptible to biodegradation by endodontically relevant microbes by a method known to show RS degradation. Emulsions of RS were dispersed in agar with minimal bacterial nutrients in culture plates. Lipase PS served as a positive control. Pseudomonas aeruginosa, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas asaccharolytica, Enterococcus faecalis, Streptococcus sanguis, Streptococcus mutans, Staphylococcus aureus, and Staphylococcus epidermidis were tested for their ability to biodegrade RS. The bacteria were inoculated in the plates and examined daily for RS degradation for 7 days. Degradation of the emulsified RS manifested in the formation of clear zones around P aeruginosa, P intermedia, P asaccharolytica, S aureus, and S epidermidis. No degradation was seen with the other tested bacteria or in plates that did not contain RS emulsion. Endodontic pathogenic bacteria can degrade RS. These findings complement other work and suggest that the seal and integrity of root canal fillings obturated with RS may be impaired by a microbial insult.

Antibacterial Activity of Cinnamaldehyde and Estragole Extracted from Plant Essential Oils against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker disease in kiwifruit. Antibacterial activity of plant essential oils (PEOs) originating from 49 plant species were tested against Psa by a vapor diffusion and a liquid culture assays. The five PEOs from Pimenta racemosa, P. dioica, Melaleuca linariifolia, M. cajuputii, and Cinnamomum cassia efficiently inhibited Psa growth by either assays. Among their major components, estragole, eugenol, and methyl eugenol showed significant antibacterial activity by only the liquid culture assay, while cinnamaldehyde exhibited antibacterial activity by both assays. The minimum inhibitory concentrations (MICs) of estragole and cinnamaldehyde by the liquid culture assay were 1,250 and 2,500 ppm, respectively. The MIC of cinnamaldehyde by the vapor diffusion assay was 5,000 ppm. Based on the formation of clear zones or the decrease of optical density caused by these compounds, they might kill the bacterial cells and this feature might be useful for managing the bacterial canker disease in kiwifruit.

Impact of Pre- and Post–Harvest Applications of Natural Antimicrobial Products on Apple and Pear Soft Rot Disease

Three natural antibacterial compounds including bacteriocin like substance (BLS) produced from lactic acid bacteria (LAB), ethanolic extract of propolis (EEP), and nine plant extracts were evaluated against soft rot Bacillus strains. Testing in vivo these compounds were evaluated to control pear and apple soft rot disease. Among eight BLS tested, BLS of LAB2, LAB105 and LAB 107 exhibited the highest antibacterial activity as indicated by the formation of clear inhibition zone. Propolis extracts exhibited significant antibacterial activity against all tested soft rot Bacillus strains and it was noticed that the antibacterial activity was concentration dependent. Among nine plant extracts tested, extracts of Eucalyptus globulus and Psidium guajava exhibited the highest antibacterial activity. All tested antibacterial products significantly decreased apple and pear soft rot severity caused by Bacillus altitudinis compared to untreated control. The highest reduction percentage of soft rot severity was recorded for EEP followed by BLS from LAB and plant extracts tested, respectively. Combined pre-and post-harvest treatments of apple and pear with antimicrobial compounds proved to be more effective in reducing the soft rot severity and improved the physical and chemical properties of fruits during storage in both years of the study. The natural antimicrobial agents used in this study were promising compounds, since it seems to be more safe, economical and great potential for extending the shelf life and improve the quality of fruits. Therefore, the application of these compounds in the control of apple and pear soft rot could be advantageous for consumers, producers, and the environment.

Selection of Potential Fungi for Production of Cellulase-Poor Xylanase from Rice Straw

Recently, xylanase has attracted attention in the pulp and paper industry because of its bleach-boosting properties, which reduces chemical consumption during the bleaching process. Four fungi species, namely Penicillium oxalicum T3.3, Aspergillus niger ATCC 6275, Colletotrichum gloeosporioides, and Pycnoporus sanguineus, were studied to select which fungi can best produce cellulase-poor xylanase using rice straw as a substrate. The fungi were screened for xylanase production based on the clear zone formation on a malt extract agar (MEA) plate containing xylan. P. oxalicum T3.3 and A. niger ATCC 6275 showed greater clear zone formation on the agar plate than the other two studied fungi. These fungi were grown in a liquid medium containing rice straw as the substrate. P. oxalicum T3.3 showed the highest xylanase activity (65.89 U/mL) with lowest carboxymethyl cellulase (CMCase) (1.88 U/mL) and filter paperase (FPase) activity (0.16 U/mL) after four days of fermentation at 30 °C. P. oxalicum T3.3 produced higher xylanase activity when rice straw was used as a substrate compared to commercial xylan. Thus, cellulase-poor xylanase produced from P. oxalicum T3.3 has high potential for biobleaching application in the pulp and paper industry in terms of technical and biological performance and economical aspects.

Characterization and Identification of Cellulolytic Bacteria from gut of Worker Macrotermes gilvus

As a social insect, termite colony consists of three castes, i.e. reproductive, soldier, and worker castes. In their role of cellulose digestion, the worker termites use two sources of cellulolytic enzyme that include cellulases produced by the termite and the gut symbions. Macrotermes gilvus classified in mound builder termite, mostly depend on cellulolytic bacteria for cellulose digestion. This study aims to characterize cellulolytic bacteria of termite gut symbionts of worker M. gilvus and to identify the cellulolytic bacteria based on sequences of 16S ribosomal RNA (rRNA) gene. Cellulolytic bacteria of termite gut were isolated and cultured in CMC (Carboxymethyl cellulose) media. The biochemical characters of bacterial isolates were assayed using Microbact 12A and 12B. Cellulolytic activity was determined based on formation of clear zone and cellulolytic index on CMC plate media. The bacterial isolate that has the highest cellulolytic index was analyzed for its 16S rRNA gene sequences. Four isolates of cellulolytic bacteria were successfully isolated from gut of M. gilvus with aerobic and anaerobic conditions. The highest formation of cellulolytic index (2.5) was revealed by RA2. BLAST-N (Basic Local Alignment Search Tool for Nucleotides) result of 16S rRNA gene sequences of RU4 and RA2 isolates showed that the isolate has similarity with Bacillus megaterium and Paracoccus yeei, respectively. This result indicated that RA2 isolate was P. yeei, a cellulolytic bacterium of a termite gut of M. gilvus.

Evaluation of Cytotoxicity, Antimicrobial and Antioxidant Enzyme Activity of Diploid and Tetraploid Platycodon grandiflorum

This experiment was conducted to obtain the have higher contents of pharmaceutical constituents as well as higher yield from colchicine induced diploid and tetraploid extracts of Platycodon grandiflorum. In order to determine the biological activity, this study was focused to evaluate the cytotoxicity, antimicrobial on the bronthus disease bacteria, antioxidant enzyme activity of diploid and tetraploid extracts in P. grandiflorum. The activities of antioxidant enzyme according to different solvent extracts were measured as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX). The cytotoxicity of methanol extracts of P. grandiflorum showed significant differences between tetraploid and diploid. That is, the cytotoxic effect against human cancer cell was higher in tetraploid than in diploid. At all extracts concentration, tetraploid samples showed high toxicity and the <TEX>$IC_{50}$</TEX> (concentration causing 50% cell death) value showed the highest on HCT-116 cell (<TEX>$105.91{\mu}g/mL$</TEX>), and exhibited significant activity against the Hep 3B cell (<TEX>$140.67{\mu}g/mL$</TEX>), SNU-1066 cell (<TEX>$154.01{\mu}g/mL$</TEX>), Hela cell (<TEX>$158.37{\mu}g/mL$</TEX>), SNU-601 cell (<TEX>$182.67{\mu}g/mL$</TEX>), Calu-6 cell (<TEX>$190.42{\mu}g/mL$</TEX>), MCF-7 cell (<TEX>$510.19{\mu}g/mL$</TEX>). Antimicrobial activities of diploid P. grandiflorum were relatively low compared to tetraploid P. grandiflorum on most of the bacterial strains. In tetraploid P. grandiflorum, K. pneumoniae showed the clear zone formation (18~19 mm) of growth inhibition, followed by the clear zone formation of 13~15 mm on C. diphtheria and S. pyogenes. The antimicrobial activities in diploid P. grandiflorum were the highest on K. pneumonia (14~15 mm), and showed the clear zone formation of 11~12 mm on C. diphtheria and 12~13 mm on S. pyogenes. The antimicrobial activity is thought to look different depending on the bacterial strains and the polyploidy of P. grandiflorum. The root extract of P. grandiflorum had the highest (97.2%) SOD enzyme activity in ethyl acetate partition layer of tetraploid while water partition layer of diploid showed the lowest (48.6%) SOD enzyme activity. The activity of CAT showed higher values in the root of tetraploid than in the diploid of P. grandiflorum in all partition layers except butyl alcohol. The activities of APX and POD showed higher values in the root of tetraploid than in the diploid of P. grandiflorum in all fraction solvents except water layer. These results indicate that the tetraploid P. grandiflorum can be used as a source for developing cytotoxic agent and antimicrobials which can act against bronchus diseases bacterial strains.

Isolation and screening of lipolytic fungi from coastal waters of the southern caspian sea (north of iran).

Background:Lipases (acylglycerol acylhydrolase, E. C. 3. 1. 1. 3) are widely distributed among microorganisms, animals and plants, catalyzing the hydrolysis of triglycerides to free fatty acids and glycerol. Their commercial application includes pharmaceutical, chemical, and paper industries.Objectives:This study aimed to isolate and screen lipolytic fungi from coastal waters of the southern Caspian Sea by Internal Transcribed Spacer-Polymerase Chain Reaction (ITS-PCR), and to optimize their lipolytic activity, pH and temperature. The ITS regions possess a high variation among taxonomically distinct fungal species and even within species.Materials and Methods:All fungal were tested to determine their lipolytic activity by the Tributyrin agar plate assay. After DNA extraction, lipase-producing fungi were identified via ITS-PCR of rDNA region with ITS1 and ITS4 primers.Results:Four fungal species were isolated from water samples of the Caspian Sea (north of Iran) between February and June 2011. The nucleotide sequences reported for three of these isolates have been assigned accession numbers from NCBI Gene Bank database. Among these species, Cladosporium langeronii showed maximum lipolytic activity (34 U/mL) and maximum clear zone formation (6 mm) on the Tributyrin agar plates. The optimum pH and temperature for activity were 8.0 and 35°C, respectively.Conclusions:The findings of this study indicated that these isolates were plant pathogenic fungi, which entered seawater from the environment, and were likely to have a suitable lipase activity on plant oils.

Clear Zone Formation around Screws in the Early Postoperative Stages after Posterior Lumbar Fusion Using the Cortical Bone Trajectory Technique.

Study DesignRetrospective study.PurposeTo evaluate the initial fixation using the cortical bone trajectory (CBT) technique for posterior lumbar fusion through assessment of the clear zones around the screws and the risk factors involved.Overview of LiteraturePostoperative radiolucent zones (clear zones) are an indicator of poor conventional pedicle screw fixation.MethodsBetween January 2013 and April 2014, 19 patients (8 men and 11 women) underwent posterior lumbar interbody fusion or posterior lumbar fusion using the CBT technique. A total of 109 screws were used for evaluation with measurement of the maximum insertional torque of last two screw rotations. Clear zone-positivity on plain radiographs was investigated 6 months after surgery. The relation between intraoperative insertional torque and clear zone-positivity was investigated by one-way analysis of variance. In addition, the correlation between clear zone-positivity and gender, age (<75 years old or >75 years old), or operative stabilization level (<2 or >3 vertebral levels) was evaluated using the chi-square test.ResultsClear zones were observed around six screws (5.50%) in five patients (26.3%). The mean insertional torque (4.00±2.09 inlbs) of clear zone-positive screws was lower than that of clear zone-negative screws (8.12±0.50 in-lbs), but the difference was not significant. There was a significant correlation between clear zone-positivity and operative level of stabilization.ConclusionsThe low incidence of clear zone-positive screws indicates good initial fixation using the CBT technique. Multilevel fusions may be risk factors for clear zone generation.

The pollen tube clear zone: clues to the mechanism of polarized growth.

Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplasmic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.

Effective Antimicrobial Filter From Electrospun Polyacrylonitrile-silver Composite Nanofibers Membrane For Conductive Environments

Electrospun nanofibers based antimicrobial filter were examined for their capability to build conductive environment. An antimicrobial agent, silver nitrate (AgNO3), was added to the nanofibers membrane for its ability to prevent growth of microorganisms over the filter media. In this direction in the present investigation the different fractions of silver nanoparticles were in-situ synthesized in PAN solution and then polyacrylonitrile (PAN)-silver composite nanofibers membrane filter was prepared by electrospinning technique. The resultant solution and PAN-silver composite nanofibers was characterized by UV- visible spectroscopy, scanning electron microscope, atomic force microscope and X-ray diffraction. Antibacterial property of PAN silver composite nanofibers were investigated against gram positive Staphylococcus aureus and gram negative Escherichia coli microorganisms. The formation of clear zone suggests that composite nanofibers containing silver nanoparticles show strong antibacterial activity and it increases with increasing silver content in the composite nanofibers. The PAN-silver composite nanofibers sheet was also examined for filtration of microorganisms and dust particles. It was observed that PAN-silver composite nanofibers filter proven to be an excellent filter for creating microorganism and dust free hygienic environment. Thus electrospun PAN nanofibers filters containing an antibacterial agent can be a promising solution for effective microorganism filtration from indoor air in hospitals or other places which are more prone to bacterial infections. Copyright © 2014 VBRI press.

Identification of new rubber-degrading bacterial strains from aged latex

Rubber waste management plays an important role to deal with high production and consumption of rubber. In order to find an alternative for current rubber waste management, rubber-degrading bacteria were isolated from aged latex. Among the 13 isolates obtained, isolate CFMR-7 showed clear zone formation around its colonies on the latex overlay agar. Phase-contrast light microscope and scanning electron microscope (SEM) analysis showed that this strain contains branched conidial filaments. Besides, CFMR-7 showed its ability to degrade latex glove whereby, increment of dry biomass weight, increase in roughness of latex glove surface and bacterial colonization on the latex glove. Analysis of 16S rRNA gene sequence revealed that CFMR-7 is 99% identical to Streptomyces sp. whereas isolates CDRG-7 and CFR-4 are 99% identical to Gordonia sp. Subsequently, partial fragment of putative latex clearing protein (lcp) gene was successfully amplified from Streptomyces sp. CFMR-7, Gordonia sp. CDRG-7 and Gordonia sp. CFR-4. Amplified DNA fragment from Streptomyces sp. CFMR-7 exhibited 81% identity to both Streptomyces sp. K30 and Streptomyces sp. LCIC4 lcp gene, whereas those from Gordonia sp. CDRG-7 and Gordonia sp. CFR-4 showed 99% and 78% identity to lcp gene of Gordonia polyisoprenivorans VH2 and Gordonia westfalica Kb1 respectively. Streptomyces sp. CFMR-7 was subjected to whole genome sequencing and full length lcp gene with 1242 bp was obtained. It showed only 77% identity to the nucleotide sequence and 73% identity to the amino acid sequence of the reported lcp gene from rubber-degrading Streptomyces. Analysis of the sequences shows that Streptomyces sp. CFMR-7 and Gordonia sp. CFR-4 are different from the reported rubber-degrading bacteria, suggesting further characterization and exploitation in rubber waste management.

Phosphate Solubilizing Bacillus megaterium mj1212 Regulates Endogenous Plant Carbohydrates and Amino Acids Contents to Promote Mustard Plant Growth.

The current study was conducted to explore the potential of a phosphate solubilizing soil bacterium, Bacillus megaterium mj1212 for enhancing the growth of mustard plants. The newly isolated bacterial strain mj1212 was identified as B. megaterium using phylogenetic analysis and, its phosphate solubilization ability was shown by the clear zone formation on National Botanical Research Institute's Phosphate medium. Moreover, the phosphate solubilization ability of B. megaterium mj1212 was enhanced by optimal culture conditions at pH 7.0 and 35°C which might be due to the presence of malic and quinic acid in the culture medium. The beneficial effect of B. megaterium mj1212 in mustard plants was determined by an increasing shoot length, root length and fresh weight of plants. In the biochemical analysis revealed that chlorophyll, sucrose, glucose, fructose and amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ilu, Leu, Tyr, Phe, Lys, His, Arg and Pro) were higher in B. megaterium mj1212 treated plants, when compared to their control. The result of present study suggests that B. megaterium mj1212 treatment could be act as phosphate biofertilizer to improve the plant growth.

BIOREMEDIATION OF CHEMICAL POLLUTANTS- CONTAMINATED WATER A-BIODECOLORIZATION OF CRYSTAL VIOLET CONTAMINATED WATER BY Pseudomonas geniculata

Biodcolorization of crystal violet (C V) was investigated in aquatic system. C V decolorizing bacterium (designated isolate AT 17) was isolated from effluent sample using enrichment technique by clear zone formation. Based on morphological, physiological and 16S rDNA, this bacterium was identified as Pseudomonas geniculata. It was capable of using C V as a sole source of carbon. Nutrient agar medium inhibited C V decolorization but mineral salt medium (MSM) increased C V decolorization. C V decolorizing by P. geniculata was found to be optimum at pH 7 and 35°C. Additional carbon sources (i.e., glucose) and nitrogen sources (i.e., peptone, yeast extract and beef extract) inhibited completely C V decolorization. On the ather hand, ammonium chloride and ammonium sulphate increased the C V decolorization. Ammonium chloride was the best inorganic nitrogen source in C V decolorization. Incubation period for 7 days was the optimum for C V decolorization. P. geniculata was able to decolorize 99% of C V completely in liquid medium at pH 7 and 35 °C after 7 days comparing with uninoculated medium(control). There was no toxicity of detected C V after 7 days of incubation with P. geniculata on Bacillus subtilis as microbial bioassay test. The dissipation of C V was coinciding with increasing P. geniculata biomass in C V contaminated water. This study has shown that P.geniculata C V could be applied to remediate Chemical pollutants - contaminated water.

Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175

Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.

Antibacterial Effect of Pseudomonas sp. Biosynthesized Silver Nanoparticles on Human Pathogenic Bacteria

The present study aimed at the biosynthesis of extracellular silver nanoparticles from Pseudomonas sp. and their characterization by means of UV-visible spectrophotometer. Moreover, the study focused on the bactericidal effects of the silver nanoparticles on human pathogenic bacteria. The nanoparticles demonstrated maximum absorbance at 410 nm corresponding to the surface plasmon resonance of nano-sized materials. However, the biosynthesis of metallic nanoparticles of silver by Pseudomonas sp. was carried out through the reduction of aqueous silver ions with culture supernatants. Pathogenic bacteria being resistant to antimicrobial agents have emerged as a major health problem in recent years. In this study, the silver nanoparticles were evaluated for their role as the antibacterial agents by well diffusion method against Pseudomonas aeroginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Vibrio cholerae, and Staphylococcus aureus . The silver nanoparticles showed satisfactory results against all six pathogenic bacteria. The extent of bactericidal effects of these nanoparticles was analyzed through the formation of clear zones around the wells in the plates containing bacterial culture. Thus, the synthesis, characterization and application of biologically synthesized silver nanoparticles could present an important concern for the development of antimicrobial agents by pharmaceutical companies to control infectious diseases caused by different pathogenic bacteria.

Identification of SGS 1609 Cellulolytic Bacteria Isolated from Sargassum spec. and Characterization of The Cellulase Produced

Bacterial isolate from seaweed designated as SGS 1609 was previously found to be able to produce cellulase represented by formation of clear zone on solid medium containing carboxymethylcellulose (CMC). This research was conducted to identify the isolate and determine optimum production time as well as characterize the cellulase produced. The isolate was identified using 16s-rRNA gene analysis. Cellulase production was conducted by cultivating the isolate in the liquid medium containing CMC followed by centrifuging to get supernatant as the crude enzyme. The enzyme was then concentrated using ammonium sulfate precipitation and ultra filtration. The concentrated enzyme having higher activity produced from the concentration process was then characterized to determine its optimum pH and temperature, heat stabilization, metal ions effect and substrate specificity. The result showed that the SGS 1609 isolate was identified as Serratia marcescens with 99% similarity. The isolate produced cellulase optimally at 4 days incubation. Ultra filtration produced higher enzyme activity compared to NH4-sulfate precipitation. The enzyme concentrated by ultra filtration worked optimally at the pH of 7, temperature of 50 oC, stable at the temperature of 60 oC for 240 minutes and was increased its activity by Ca2+ and Mg2+ ions. On the other hand, the enzyme was inhibited by Fe3+, Zn2+ and Na+ ions, but was not relatively affected by K+ and EDTA. The use of conventional agar producer waste treated with 6% NaOH gave highest activity compared to other substrates.

Isolation, Identification, and Characterization of a CellulolyticBacillus amyloliquefaciensStrain SS35 from Rhinoceros Dung

Cellulose hydrolyzing bacteria were isolated from rhinoceros dung and tested for clear zone formation around the colonies on the agar plates containing the medium amended with carboxymethylcellulose as a sole carbon source. Isolates were further screened on the basis of carboxymethylcellulase production in liquid medium. Out of 36 isolates, isolate no. 35 exhibited maximum enzyme activity of 0.079 U/mL and was selected for further identification by using conventional biochemical tests and phylogenetic analyses. This was a Gram-positive, spore forming bacterium with rod-shaped cells. The isolate was identified as Bacillus amyloliquefaciens SS35 based on nucleotide homology and phylogenetic analysis using 16S rDNA and gyrase A gene sequences.

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. &lt;br /&gt;&lt;br /&gt;