Subculture Generations Research Articles

Q-PCR Methodology for Monitoring the Thermophilic Hydrogen Producers Enriched from Elephant Dung

This study aims to create a quantitative polymerase chain reaction (q-PCR) methodology for monitoring the hydrogen-producing mixed cultures enriched from elephant dung using alpha-cellulose as a carbon source through five generations of repetitive sub-culture. The enriched thermophilic mixed cultures from the fifth cultivation cycle gave the highest hydrogen yield of 170.3 mL H2/g cellulose and were used to generate hydrogen from sawdust. Clostridium sp. and Thermoanaerobacterium sp. were the dominant bacteria in thermophilic mixed cultures with high hydrogen yield, according to polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). q-PCR primers Chis150F and ClostIR, TherF and TherR, and BacdF and BacdR were developed to amplify the 16S rRNA genes of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp., respectively, for the quantification of hydrogen-producing bacteria in biohydrogen fermentation. Similar q-PCR analysis of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp. 16S rRNA gene amplification during hydrogen production from cellulose and sawdust revealed increasing gene copy number with time. The molecular approaches developed in this study can be used to monitor microbial communities in hydrogen fermentation processes efficiently.

Growth of Kaffir Lime (<i>Citrus hystrix</i> DC) Cell Line Derived from Seed Explant After Yeast Elicitation Using Pure and Technical Grade Yeast

The addition of elicitors in kaffir lime (Citrus hystrix DC.) culture is one of strategies for obtaining and increasing the production of secondary metabolites. Saccharomyces cerevisiae is one of the elicitors that can be used to increase secondary metabolites such as terpenoids. However, in its use, the pure cultures of S. cerevisiae are expensive. Therefore, the first objective of this study was to analyze the ability of technical grade (commercial baker’s yeast) to be used as an elicitor and measure the growth of kaffir lime cell line after being elicited by pure and technical grade (commercial baker’s yeast). The second objective is to determine the best time to subculture kaffir lime cell line after elicitation. We observed the morphology and measured the growth curve of pure and technical grade yeast until the 4th subculture generation. Furthermore, we used both grades of yeast for elicitation. Kaffir lime cell suspension was treated with 10 ppm pure grade or 5 ppm and 10 ppm technical grade yeast for 4 days. After elicitation, kaffir lime cell lines were subcultured and their growth was analyzed. The result showed that the morphology and growth curve of technical grade until 4th subculture generations was similar to the pure grade. On the other hand, after elicitation using pure and technical grade yeast and being subcultured, the growth of the elicitated kaffir lime cell line had the same pattern as the control group, but the cell density of the control group was higher than the elicitated group. The initial stationary phase of kaffir lime cell line was on the 17th day which is the best time to subculture. The subculturing process is important to maintain the viability of the kaffir lime cell line.

A mutant of seawater Arthrospira platensis with high polysaccharides production induced by space environment and its application potential

To improve the yields of specific value-added compounds from Arthrospira platensis, one generally accepted method is to obtain mutant strains with increased target component content and growth rate. In the present study, mutation breeding of seawater A. platensis SCSIO-44012 (the wild type strain, hereafter WT) through space-flight carriage by Shijian-10 satellite was carried out. After returning to earth, 183 single filaments were picked out, 43 of which survived. During the following consecutive subcultures, one out of the 43 mutant strains, named A. platensis SCSIO-44012-H11 (hereafter H11), showed stable advantages in high levels of polysaccharides production. The biomass yield, total sugar content, and total sugar productivity of H11 remained stable at 0.88 g L−1, 24.82% DW, and 21.78 mg L−1 d−1, respectively, from the 10th to 35th generations of subculture, which were 8.6%, 62.26%, and 73.68%, respectively, higher than that of WT. The biochemical compositions of polysaccharide extracts (PSE) from H11 and WT also exhibited significant differences. The total sugar content of H11 PSE reached 55.37%, 2.49 times as much as that of WT PSE. The PSE from H11 was enriched in the component cutting off 100 kDa, accounting for 89.32% of the total polysaccharides, whereas WT PSE was mainly composed of the component with a molecular weight between 10 kDa and 50 kDa. The predominant monosaccharide of H11 PSE was glucose, followed by rhamnose, which accounted for 84.53% and 8.13% of the total monosaccharides, respectively. By comparison, glucose and rhamnose occupied 27.33% and 35.41%, respectively, in PSE from WT. Furthermore, the PSE from H11 showed strong ferrous ion-chelating activity and moisture retention capacity. These results indicated the feasibility of H11 as a polysaccharides feedstock applied in cosmetics, nutraceuticals, and medicines.

THE CONTENT OF PHENOLIC COMPOUNDS AND FLAVONOIDS IN Deschampsia antarctica TISSUE CULTURE

Aim. The aim of the study was to determine the quantitative and qualitative content of phenolic compounds and flavonoids in Deschampsia antarctica E. Desv. tissue cultures obtained from plants originating from different islands of the maritime Antarctic. Methods. In vitro tissue culture, Folin-Ciocalteu method, spectrophotometry, HPLC analysis. Results. The quantitative content of phenolic compounds and flavonoids in D. antarctica tissue cultures obtained from plants of six genotypes (DAR12, DAR13, G/D12-2a, Y66, R30 and L57) was determined. The highest content of phenolic compounds (4.46 and 3.75 mg/g) was found in tissue cultures obtained from root and leaf explants of plant genotype L57. The highest amount of flavonoids (7.17 mg/g) was accumulated in G/D12-2a tissue culture of root origin. The content of the studied biologically active compounds (BACs) did not change with increasing number of subculture generations (from passage 10 to 19). HPLC analysis showed that in D. antarctica tissue cultures, a shift in the biosynthesis of BACs occurred towards the synthesis of more polar metabolites compared to explant donor plants. Conclusions. It was found that the transition of cells to undifferentiated growth affected the content of BACs, the amount of which decreased 2–5 times simultaneously with a significant change in their profile. This provided a basis for further biochemical studies, as well as for careful selection of tissue culture of D. antarctica to use it as a potential source of BACs.

Screening of pathogenicity-deficient Fusarium oxysporum mutants established by Agrobacterium tumefaciens-mediated transformation

Apple replant disease (ARD) occurs frequently in old orchards and is an important disease that limits apple production in China. Fusarium oxysporum is one of the causal agents of ARD and its pathogenic mechanism is still unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of F. oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT system yielded 160–200 transformants/106 conidia, indicating a relatively high conversion efficiency. The genetically stable transformants were tested through five generations of successive subculture on hygromycin-free media, and the alien gene was detected via PCR. A total of 3500 transformants were obtained to produce a T-DNA insertion library of F. oxysporum HS2, and 223 of these mutants were randomly selected for sporulation and pathogenicity tests. Most of the tested mutants presented weakened virulence, and one of them displayed obviously attenuated sporulation and reduced pathogenicity. The T-DNA in six pathogenicity-deficient mutants existed as a single copy, as confirmed by Southern blotting. The T-DNA flanking sequence was obtained rapidly by successive chromosome walking via the high-efficiency thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR) method. The T-DNA right flanking sequences of five transformants were identified further, and the obtained sequences were aligned to the genomic sequence, revealing novel loci related to pathogenesis. This study provided a large number of various pathogenesis-related mutants and pathogenesis-related genes, which will be valuable resources for characterizing the molecular mechanisms of pathogenesis-related genes.

Establishment of a maize callus regeneration system from haploid shoot tips

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize. An effective haploid maize shoot tips callus regeneration system was established which enabled homozygous transgenic plants to be obtained in a single generation.

Biodegradation of Kraft Lignin using Mixed Microbial Consortia

This study presents the recent research on bacterial kraft lignin degradation. To acquire microbial consortia for successfully lignin degradation, the samples were obtained from soil source. These isolated microbial strains SB2, SB4, SB5, SB6 and SB7 were isolated and screened after generations of subculture and mixed as a co-culture. In this study mixed bacterial strain was used for lignin degradation. Maximum kraft lignin (KL) degradation was found at 35 °C with 1% of mixed seed culture on 6th day of inoculation. This study reveals that lignin degrading bacteria reducing TDS 55.81%, COD 72.64% and decolorized colour up to 48.17%. These isolated strains were playing key role in bioremediation of kraft lignin degradation and for production of chemical and biofuel. Hence, model kraft lignin can be detoxify by using mixed bacterial strains.

Improving hairy root induction of Urena lobata L. by Agrobacterium rhizogenes ATCC 15834 by some factors

Introduction: Our previous study showed that Urena lobata L. hairy root is a potential pharmaceutical source for type 2 diabetes treatment. In order to improve the transformation efficacy and the quality of hairy roots, this study examined the effects of several factors including age, parts of plants, infection time and culture medium in inducing hairy roots in Urena lobata L. 
 Methods: In this study, we investigated four factors to improve the hairy root induction in Urena lobata L. These factors include: age of plant (15-day-old in vitro plants, 45-day-old in vitro plants and after two subculture generations plants), different parts of plant (roots, stems, and leaves), infection time (10, 20 and 30 minutes), and culture medium (Murashige and Skoog (MS), Gamborg B5 medium (GB5) and Woody plant medium (WPM)). All experiments were repeated three times, with uninfected leaf explants of 15-day-old in vitro as the negative control. The transformation frequency and the fresh biomass of hairy roots were recorded at four weeks after infection.
 Results: The results showed that the optimized procedure which used 15-day-old in vitro plants, the leafy part, the infection time of 10 minutes and culture in the WPM medium was better than the original procedure. The optimized procedure achieved a transformation frequency of 100%. In addition, the fresh biomass of hairy roots formed on an explant in the optimized procedure was 3.2 times higher than the ones induced by the original procedure.
 Conclusion: The results showed that the optimized procedure was more effective than the original procedure in inducing Urena lobata hairy roots.

Determination and quantification of asiaticoside in endophytic fungus from Centella asiatica (L.) Urban

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC-MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29 ± 3.36µg/100mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source. Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062).

Screening of a microbial consortium for selective degradation of lignin from tree trimmings

To acquire microbial consortia with effectively precedent degradation of lignin, samples obtained from rotten trunks, rotten stumps and soil near it were screened and isolated after generations of subculture. The dynamic change illustrated that their community structures were affected by pH and tended to be stable after 6 days’ cultivation. The desired one, named DM-1, was gained through successive cultivation for over 5 generations, whose high selectivity in lignin degradation was observed within 16 days’ cultivation (SV = 2.78). Meanwhile, a remarkable reduction in the fiber crystallinity of tree trimmings (10.35%) resulted from the bio-degradation of DM-1, displayed a greater exposure of cellulose by selective removal of lignin. The diversity analysis of DM-1 was investigated by PCR amplification and 16S rDNA sequencing, indicated that mesorhizobium, cellulosimicrobium, pandoraea, achromobacter and stenotrophomones were the predominant genera. Furthermore, fungi (3 strains), bacteria (4 strains) and actinomycetes (5 strains) constituted 12 strains in total were gained by plate isolation from DM-1.

Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture.

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

Biodegradation of Enteromorpha polysaccharides by intestinal micro-community from Siganus oramin

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL−1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.

Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography

Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum.

The investigation of epigenetic modifiers in cultures of an endophytic fungus to enhance PPT production

Podophyllotoxin (PPT) is a natural plant lignan used for the semi-synthesis of anti-cancer drugs. Due to a variety of factors, PPT is becoming less available. An endophytic fungus, PPE7, from Podophyllum peltatum, can serve as an alternate source of PPT, but production declined over several generations of subculture, perhaps due to gene silencing. Small molecule epigenetic modifiers, such as DNA methyl transferase (DNMT) and histone deacytelase (HDAC) inhibitors, can activate silent fungal pathways and enhance the production of fungal metabolites. Re-activation of the PPT pathway may make it possible to identify the genes involved in PPT synthesis through cDNA analysis. The HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) and the DNMT inhibitor 5-azacytidine (5-aza) were used to treat cultures of PPE7. Extracts were prepared by solid phase extraction (SPE) and analyzed by HPLC. Cultures treated with SAHA and 5-aza showed gain of peaks, loss of peaks and altered amounts of individual peaks compared to the untreated cultures. Thus, both epigenetic modifiers are capable of affecting the compounds produced in cultures of PPE7. LC-MS analysis is in progress to further confirm these findings and explore the production of PPT and related lignans in the treated cultures.

Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology, immunophenotype and multi-lineage potential were analyzed at passages 3, 5, 10, 15, 20, and 25 (P3, P5, P10, P15, P20, and P25, respectively). The cell cycle distribution, apoptosis, and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3, P5, P10, P15, P20, and P25. From P3 to P25, the three defining biological properties of hUC-MSCs [adherence to plastic, specific surface antigen expression, multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased, compared with the cells at P3 (P<0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183%), compared to those at P3 (P<0.01). Within subculture generations 3-20 (P3-P20), the differences between the cell apoptotic rates were not statistically significant (P>0.05). There were no detectable chromosome eliminations, displacements, or chromosomal imbalances, as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations.

Tissue culture-induced somaclonal variation of decreased pollen viability in torenia (Torenia fournieri Lind.).

BackgroundPhenotypic and genotypic variations, collectively called somaclonal variations, are induced during tissue culture.ResultsWe studied the phenotypic variation in pollen viability of regenerants of torenia after subculturing for one to nine generations. We found that pollen viability of regenerants continuously decreased with increasing subculture time. High concentrations of plant growth regulators applied to the Murashige and Skoog (MS) medium also resulted in diminished pollen viability. Furthermore, antibiotic application during gene transformation also decreased pollen viability of the transformants. However, the process of long-term culture did not significantly change pollen viability. The mean methylation level of regenerants showed a 0.28% to 3.95% decrease in seedlings subcultured in vitro for nine generations. Moreover, when the ninth subcultured regenerants with reduced pollen vibility were recovered in soil to get seeds, the pollen viability of seed-derive plants was similar to that of the wild type.ConclusionsThe results show that plant growth regulators, antibiotics, and the number of subculture generations influence somaclonal variations in torenia. The somaclonal variations in torenia may results from epigenetic changes.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-36) contains supplementary material, which is available to authorized users.

Construction of a controllable β‐carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable β-carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80-100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β-carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae.

PCR-based RAPD technique to determine induced salinity tolerance in vitro in Acacia auriculiformis

Acacia auriculiformis is an agro-forestry species in India with application in paper industry. The species is saline intolerant and fails to establish. Experiments were conducted to induce saline tolerance in callus cultures of A. auriculiformis on Murashige & Skoogs medium supplemented with different concentrations of NaCl+CaCl2––– (2:1; 50, 100, 150, 200, 250 or 300 mM). Calli were trained for eight sub-culture generations. The genome from saline-tolerant calli was extracted by modified cetyl trimethyl ammonium bromide method and subjected to polymerase chain reaction-based random amplified polymorphic DNA technique. The polymorphic DNA bands were eluted and cloned to Escherichia coli with the help of pTZ57R/T. The transformed plasmid DNA was sequenced and subjected to BLAST at National Center for Biotechnology Information. Results indicated that saline training of calli resulted in the expression of tolerance to 100 and 150 mM saline concentration after eight sub-cultures. Both the saline concentrations produced conspicuous bands in trained calli. Three nucleotide sequences were similar to those of Bacillus amyloliquefaciens sub sp. plantarum and BLAST search result indicated high homologies to ThiT thiamine transporter, YuaG flottilin type band 7, and U32 class peptidase protein genes in bacterial genome. The annotation of these protein genes in saline tolerance was discussed. The study suggested that the endophytic B. amyloliquefaciens sub sp. plantarum might be inducing saline tolerance in A. auriculiformis.

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC.In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production.

Stability of micropropagated Musa acuminata cv. Grand Naine over clonal generations: A molecular assessment

The present work was undertaken to investigate clonal fidelity of banana ( Musa acuminata cv. Grand Naine) regenerants from six different in vitro subculture generations and in the explant suckers by using ISSR and REMAP molecular markers. Both types of markers revealed high degree of monomorphism. Very low variation was observed up to the eighth subculture generation with polymorphic bands being low in both ISSR (0.96%) and REMAP (0.95%) markers. Epigenetic stability was studied by DNA methylation analysis of the eighth subculture generation samples. Single 570 bp methylation sensitive band was absent in two of the fifteen MspI predigested samples, while it was present in HpaII predigested and undigested samples. The results of the investigation confirmed that the micropropagation of banana up to the eighth subculture generation show low variation.