Atit Kanti

An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species.

Natural Product Libraries to Accelerate the High Throughput Discovery of Therapeutic Leads

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.

Pichia chibodasensis sp. nov., isolated in Indonesia

Three strains (14Y260T, 14Y268 and 14Y276) of xylose-assimilating yeasts were isolated from decayed wood and soil collected in West Java in Indonesia. A phylogenetic analysis was performed based on the sequences of the D1/D2 domains of LSU, SSU and EF-1α, and the three strains were found to belong to the genus Pichia. The morphological, biochemical, physiological and chemotaxonomic characteristics indicated that these strains were distinct from other closely related species. Strains 14Y260T, 14Y268 and 14Y276 belonged to the Pichia clade and represent a novel species, named Pichia chibodasensis sp. nov. ; The type strain is 14Y260T (=NBRC 111569T=InaCC Y1042T).

Lipid Accumulation By ATH Using Palm Oil Mill Effluent As Substrate

Large amount of palm oil mill effluent (POME) is generated annually. The waste would bepotential for production of single cell oils (SCOs). The objective of this study was to evaluatelipid accumulation by fungi using POME as substrate. Seven filamentous fungi were initiallyisolated from various biomes. The study results showed that out of these 7 fungi, five of themproduced endoglucanase and accumulated lipid about 34.3-87.5 persen of their dry cell mass usingPOME as substrate. The five fungi were identified as ATH, sp.,sp., sp. 1 T30, and sp.2 T50. The highest lipid accumulation wasobtained by ATH. The profile of trans-esterified SCOs revealed high content ofsaturated and mono-unsaturated fatty acids i.e., palmitic (C16:0), stearic (C18:0) and oleic(C18:1) acids similar to conventional vegetable oils used for biodiesel production. The strainthat was able to use organic substrates in POME indicated that they are promising strain forbiofuel feedstock as well as for fulfilling effluent quality for wastewater discharge BIOTROPIA Vol. 21 No. 2, 2014: 100 - 110

Diversity of Ethanol Producing Yeasts Isolated from Fermented Foods in Riau Islands

Information on genetic diversity of fermentative yeast which produce ethanol is very crucial in developing biofuel production in Indonesia. Research on ethanol producing yeasts is interest of many scientist. The objective of study was to reveal yeast diversity in Indonesian fermented foods that able to produce ethanol. The sample of fermented foods were collected in the traditional market in Karimun Besar Island, Kepulauan Riau. Yeast isolation was performed using serial dilution with direct plating and enrichment culture with glucose as carbon source. Fifteen of isolates were isolated and identified by amplification of D1/D2 region LSU 26S rDNA. Its ethanol production characteristic was analyzed base on fermentation activity and measurement with gas chromatography for ethanol content. The result revealed that 8 yeast species were found belong to Ascomycetous and grouped into 5 clades which are able to produce ethanol. The highest ethanol production was obtained by Saccharomyces cerevisiae Y15Kr107 (3.53 persen) followed by Torulaspora delbrueckii Y15Kr104 (1.63 persen), Saccharomyces cerevisiae Y15Kr093 (1.58 persen), Candida glabrata Y15Kr110 (1.4 persen), Torulaspora delbrueckii Y15Kr103 (1.29 persen), Candida glabrata Y15Kr108 (1 persen), Torulaspora globosa Y15Kr094 (0.92 persen), Kodamaea ohmeri Y15Kr096 (0.61 persen), and Pichia kudriavsevii Y15Kr106 (0.31 persen) Y15Kr105 (0.21 persen) Y15Kr109 (0.16 persen). Other yeasts strains did not produce ethanol but may play different role in fermentation process. Jurnal Biologi Indonesia, 2017, Vol 13(1), 61-69

Keragaman Jenis Khamir Penghasil Etanol yang Diisolasi dari Makanan Fermentasi di Kepulauan Riau

ABSTRACT Information on genetic diversity of fermentative yeast which produce ethanol is very crucial in developing biofuel production in Indonesia. Research on ethanol producing yeasts is interest of many scientist. The objective of study was to reveal yeast diversity in Indonesian fermented foods that able to produce ethanol. The sample of fermented foods were collected in the traditional market in Karimun Besar Island, Kepulauan Riau. Yeast isolation was performed using serial dilution with direct plating and enrichment culture with glucose as carbon source. Fifteen of isolates were isolated and identified by amplification of D1/D2 region LSU 26S rDNA. Its ethanol production characteristic was analyzed base on fermentation activity and measurement with gas chromatography for ethanol content. The result revealed that 8 yeast species were found belong to Ascomycetous and grouped into 5 clades which are able to produce ethanol. The highest ethanol production was obtained by Saccharomyces cerevisiae Y15Kr107 (3.53%) followed by Torulaspora delbrueckii Y15Kr104 (1.63%), Saccharomyces cerevisiae Y15Kr093 (1.58%), Candida glabrata Y15Kr110 (1.4%), Torulaspora delbrueckii Y15Kr103 (1.29%), Candida glabrata Y15Kr108 (1%), Torulaspora globosa Y15Kr094 (0.92%), Kodamaea ohmeri Y15Kr096 (0.61%), and Pichia kudriavsevii Y15Kr106 (0.31%) Y15Kr105 (0.21%) Y15Kr109 (0.16%). Other yeasts strains did not produce ethanol but may play different role in fermentation process. Key words: yeast, fermented food, ethanol, Kepulauan Riau

Three novel species of d-xylose-assimilating yeasts, Barnettozyma xylosiphila sp. nov., Barnettozyma xylosica sp. nov. and Wickerhamomyces xylosivorus f.a., sp. nov.

This study describes three novel xylose-assimilating yeasts, which were isolated from decayed wood collected from Bung Hatta Botanical Garden in West Sumatra and Cibodas Botanic Garden in West Java, or from litter from Eka Karya Bali Botanic Garden in Bali, Indonesia. Phylogenetic analysis was performed based on the sequences of the D1/D2 domains of the large ribosomal subunit (LSU), the small ribosomal subunit (SSU), the internal transcribed spacer (ITS) and elongation factor-1α (EF-1α), and the three strains were found to represent three novel species belonging to genera Barnettozyma or Wickerhamomyces. The morphological, biochemical and physiological characteristics indicated that the strains were distinct from other closely related species. Strains 13Y206T and 14Y196T belonging to the Barnettozyma clade are described as the type strains of Barnettozyma xylosiphila sp. nov. (type strain 13Y206T=NBRC 110202T=InaCC Y726T; MycoBank MB808598) and Barnettozyma xylosica sp. nov. (type strain 14Y196T=NBRC 111558T=InaCC Y1030T; MycoBank MB819485). Strain 14Y125T belonging to the Wickerhamomyces clade is described as the type strain of Wickerhamomyces xylosivorus f.a., sp. nov. (type strain 14Y125T=NBRC 111553T=InaCC Y1026T; MycoBank MB819484).

KEANEKARAGAMAN KHAMIR YANG DIISOLASI DARI SUMBER DAYA ALAM PULAU ENGGANO, BENGKULU DAN POTENSINYA SEBAGAI PENDEGRADASI SELULOSA

The present study revealed the occurrence and diversity of yeasts from natural resources of Enggano Island. The yeasts were isolated from soil, sediment, leaf, leaf litter, fruit, and flower. Isolation was performed using direct plating, membrane filtration, ballistospore-falling and enrichment culture using glucose, xylose and xylan as carbon source. After isolating, yeasts were screened for its cellulolytic potency. The occurrence of yeasts in different isolation technique are clearly found. Most of 87 yeasts isolated, abundant in leaf, soil, and sediment sample. The total yeasts isolated were classified into 32 species based on D1/D2 LSU 26S rDNA sequences analysis which genera of Candida were predominant (33%). The predominant yeast species were identified as Candida tropicalis (16,1%), Cyberlindnera saturnus (16,1%), and Rhodosporidium paludigenum (11,5%). Screening on carboxymethyl cellulose medium, there are 43 isolates within 22 yeasts species have cellulolytic activity. Four species of Basidiomycetous have high activity which includes Pseudozyma antartica Y15Eg001; Pseudozyma hubeiensis Y15Eg015; Anthracocystis chrysopogonis Y15Eg072; Cryptococcus laurentii Y15Eg017 and two from Ascomycetous are Sarocladium bactrocephalum Y15Eg226, Y15Eg227. Some of cellulolytic yeast isolates are indicated as new species candidate and required to reconfirm in another conserve regions to ensure its taxonomic position. Various yeasts isolated from Enggano Island with its cellulolytic potency should contribute to scientific information regarding microbial genetic resources of outer islands of Indonesia.

DIVERSITAS DAN PROFIL METABOLIT SEKUNDER JAMUR ENDOFIT YANG DIISOLASI DARI TUMBUHAN GAMBIR (Uncaria gambler) SERTA AKTIVITAS BIOLOGISNYA SEBAGAI ANTIBAKTERI [Diversity and Secondary Metabolites Profiles of Endophytic Fungi Isolated from Gambir {Uncaria gambier) Plants and Their Biological Activities as Antibacteria]

Five kinds of endophytic filamentous fungi i.e. Pestalotiopsis sp. GNC, Fusarium sp. GNC-A, Fusahum sp. GNC-B, Pestalotiopsis sp. GUC and Fusarium sp. GUC were isolated from surface sterilized of steems of gambir nasi and gambir udang (Uncaria gambier Roxb.) collected from Rengat, Riau Province,Indonesia. Identification of the endophytic fungi were done through morphologically observations and chemotaxonomically approach. In an antibacterial assays, the ethyl acetate extract of the Pestalotiopsis sp.GNC cultures showed their activity against pathogenic bacteria, Eschericia coli, Staphylococcus aureus and Bacillus sublilis.Beside the above three pathogenic bacteria, the ethyl acetate extract of Fusarium sp. GNC-B culture was also active against