Efficient Production of Lactic Acid from Sweet Sorghum Juice by a Newly Isolated Lactobacillus salivarius CGMCC 7.75

Sweet sorghum juice was a cheap and renewable resource, and also a potential carbon source for the fermentation production of lactic acid (LA) by a lactic acid bacterium. One newly isolated strain Lactobacillus salivarius CGMCC 7.75 showed the ability to produce the highest yield and optical purity of LA from sweet sorghum juice. Studies of feeding different concentrations of sweet sorghum juice and nitrogen source suggested the optimal concentrations of fermentation were 325 ml l⁻¹ and 20 g l⁻¹, respectively. This combination produced 142.49 g l⁻¹ LA with a productivity level of 0.90 g of LA per gram of sugars consumed. The results indicated the high LA concentration achieved using L. salivarius CGMCC 7.75 not only gives cheap industrial product, but also broaden the application of sweet sorghum.

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The online version of this article (doi:10.1007/s12088-013-0377-0) contains supplementary material, which is available to authorized users.     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Enhanced Glucosamine Production with Actinomucor elegans Based on Stimulating Factor of Methanol

Glucosamine (GlcN) is a major and valuable component in the cell wall of fungi. In this study, the cell wall was treated via a two-stage alkali and acid process, and chitin and chitosan were fully deacetylated, partially depolymerized, and converted to GlcN oligosaccharides. Then, the oligosaccharides were analyzed by high performance liquid chromatography. The influences of Actinomucor elegans on GlcN production in a flask culture were investigated to achieve an optimum yield of GlcN. The experimental result showed that cultivation in condition of pH 6.0, 100 mL working volume (500 mL flask), 10 % (v/v) inoculum concentration, at 28 °C and 200 rpm for 6 days yielded highest dry cell weight (DCW) which was 23.43 g L⁻¹, with a GlcN concentration of 5.12 g L⁻¹. Methanol as stimulating factor was found to exert the best effect in concentration of 1.5 % (v/v). With addition of methanol into medium, the DCW increased from 23.69 to 32.42 g L⁻¹, leading to maximum GlcN concentration of 6.85 g L⁻¹ obtained. Here, the methanol addition may be useful for industrial production of GlcN, and may also be meaningful for the production of other fine chemicals by filamentous fungi.     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L⁻¹ yeastolate ultrafiltrate, 10 g L⁻¹ glucose, 3.5 g L⁻¹ glutamine, 0.5 g L⁻¹ fructose, 2 g L⁻¹ lactose, 0.6 g L⁻¹ tyrosine, 1.48 g L⁻¹ methionine and 1% (v/v) lipid emulsion, reaching 19 × 10⁶ cells mL⁻¹. Maximum specific growth rate and cell productivity were 0.025 h⁻¹ and 0.57 × 10⁵ cells mL⁻¹ h⁻¹, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L⁻¹, however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.     

Micropropagation of Hedychium coronarium J. Koenig through rhizome bud

An optimized protocol was developed for in vitro plant regeneration of a medicinally important herb Hedychium coronarium J. Koenig using sprouted buds of rhizomes. The rhizomes with sprouted bud were inoculated on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) supplemented with either N⁶-benzyladenine (BA) alone (1.0–4.0 mg L⁻¹) or in combination with 0.5 mg L⁻¹ naphthalene acetic acid (NAA). Of these combinations, MS supplemented with a combination of 2.0 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA was most effective. In this medium, best shoots (3.6) and roots (4.0) regeneration was observed simultaneously with an average shoot and root length of 4.7 cm and 4.2 cm respectively. Regeneration of shoots and roots in the same medium at the same time (One step shoot and root regeneration) reduced the time for production of in vitro plantlets and eliminates the media cost of rooting. Cent-percent (100 %) success in plant establishment was observed in both gradual acclimatization process as well as when plants were directly transferred to outdoor in clay pots containing a mixture of garden soil and sand (2:1) without any sequential acclimatization stage.     

An efficient and reproducible indirect shoot regeneration from female leaf explants of Simmondsia chinensis,a liquid-wax producing shrub

Simmondsia chinensis (Link) Schneider is a perennial, dioecious, drought resistant and multipurpose seed oil crop grown in arid and semi-arid conditions throughout the world. A reproducible and more efficient method for indirect shoot organogenesis from female leaf explants has been standardized. The leaf explants cultured on Murashige and Skoog (MS) medium with 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone produced the highest frequency of callus compared with 1.5 mg l⁻¹ IBA. Maximum proliferation of callus was observed on MS medium containing a combination of 1.0 mg l⁻¹ 2,4-D with 0.5 mg l⁻¹ BAP. For shoot differentiation, the proliferated callus was subcultured on MS medium supplemented with 6-benzylaminopurine (BAP) (1.0–4.0 mg l⁻¹) along with 40 mg l⁻¹ adenine sulphate as additive or in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA). Optimum shoots differentiated from callus was obtained on MS medium supplemented with 2.0 mg l⁻¹ BAP and 0.2 mg l⁻¹ NAA. On this medium, 100 % cultures were responded with an average number of 14.44 shoots per explant with their mean length of 4.78 cm. In vitro rooting (6.22 roots per explant) was achieved on half strength MS medium containing 2 % sucrose with 3.0 mg l⁻¹ IBA and 300 mg l⁻¹ activated charcoal (AC). Rooted plantlets were successfully hardened under control conditions and acclimatized under field conditions with 90 % success rate. The present protocol is highly efficient, reproducible and economically viable for large scale production of female plants.     

Shoot culture of Bacopa monnieri: standardization of explant,vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5 mg l⁻¹) and IAA (0.01 mg l⁻¹) with 3 % sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5 cm explant size and 20 explant/40 ml (1 explant/2 ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100 ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250 ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l⁻¹ medium as compared to control (938 shoots l⁻¹). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.     

Role of nitrogen and magnesium for growth,yield and nutritional quality of radish

Nitrogen (N) affects all levels of plant function from metabolism to resource allocation, growth, and development and Magnesium (Mg) is a macronutrient that is necessary to both plant growth and health. Radish (Raphanus sativus L.) occupies an important position in the production and consumption of vegetables globally, but there are still many problems and challenges in its nutrient management. A pot trial was conducted to investigate the effects of nitrogen and magnesium fertilizers on radish during the year 2018–2019. Nitrogen and magnesium was applied at three rates (0, 0.200, and 0.300 g N kg⁻¹ soil) and (0, 0.050, and 0.100 g Mg kg⁻¹ soil) respectively. The experiment was laid out in a completely randomized design (CRD) and each treatment was replicated three times. Growth, yield and quality indicators of radish (plant height, root length, shoot length, plant weight, total soluble sugar, ascorbic acid, total soluble protein, crude fiber, etc.) were studied. The results indicated that different rates of nitrogen and magnesium fertilizer not only influence the growth dynamics and yields but also enhances radish quality. The results revealed that the growth, yield and nutrient contents of radish were increased at a range of 0.00 g N. kg⁻¹ soil to 0.300 g N. kg⁻¹ soil and 0.00 g Mg. kg⁻¹ soil to 0.050 g Mg. kg⁻¹ soil and then decreased gradually at a level of 0.100 g Mg. kg⁻¹ soil. In contrast, the crude fiber contents in radish decreased significantly with increasing nitrogen and magnesium level but increased significantly at Mg₂ level (0.050 g Mg. kg⁻¹ soil). The current study produced helpful results for increasing radish quality, decreasing production costs, and diminishing underground water contamination.     

Plant Growth Promoting Bacteria from Cow Dung Based Biodynamic Preparations

Indigenous formulations based on cow dung fermentation are commonly used in organic farming. Three biodynamic preparations viz., Panchagavya (PG), BD500 and ‘Cow pat pit’ (CPP) showed high counts of lactobacilli (10⁹ ml⁻¹) and yeasts (10⁴ ml⁻¹). Actinomycetes were present only in CPP (10⁴ ml⁻¹) and absent in the other two. Seven bacterial isolates from these ferments were identified by a polyphasic approach: Bacillus safensis (PG1), Bacillus cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysinibacillus xylanilyticus (BD3) and Bacillus licheniformis (CPP1). This is the first report of L. xylanilyticus and B. licheniformis in biodynamic preparations. Only three carbon sources—dextrose, sucrose and trehalose out of 21 tested were utilized by all the bacteria. None could utilize arabinose, dulcitol, galactose, inositol, inulin, melibiose, raffinose, rhamnose and sorbitol. All the strains produced indole acetic acid (1.8–3.7 μg ml⁻¹ culture filtrate) and ammonia. None could fix nitrogen; but all except B. safensis and B. licheniformis could solubilize phosphorous from insoluble tri-calcium phosphate. All the strains except L. xylaniliticus exhibited antagonism to the plant pathogen Rhizoctonia bataticola whereas none could inhibit Sclerotium rolfsi. In green house experiment in soil microcosms, bacterial inoculation significantly promoted growth of maize; plant dry weight increased by ~21 % due to inoculation with B. cereus (PG2). Results provide a basis for understanding the beneficial effects of biodynamic preparations and industrial deployment of the strains.

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The online version of this article (doi:10.1007/s12088-014-0468-6) contains supplementary material, which is available to authorized users.     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).     

Biodegradation of Phenanthrene by Pseudomonas putida and a Bacterial Consortium in the Presence and in the Absence of a Surfactant

This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P. putida degraded phenanthrene to form 1-hydroxy-2-naphthoic acid (1H2Na) as the major metabolite. LC–MS analysis revealed the production of complementary intermediates in the presence of Brij 30, showing intense ions at mass-to-charge ratios (m/z) 97 and 195. Higher phenanthrene biodegradation rate was obtained in the presence of Brij 30. Conversely, in the case of Pyr01consortium, the addition of Brij 30 (0.5 g L⁻¹) had a negative effect on biodegradation: no phenanthrene biodegradation products were detected in the medium, whereas a production of several intermediates (m/z 97, 195 and 293) was obtained without surfactant. New results on phenanthrene metabolism by P. putida DSMZ 8368 and Pyr01 consortium in the presence and in the absence of Brij 30 we obtained. They confirm that the knowledge of the effect of a surfactant on bacterial cultures is crucial for the optimization of surfactant-enhanced PAHs biodegradation.

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The online version of this article (doi:10.1007/s12088-012-0265-z) contains supplementary material, which is available to authorized users.     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l⁻¹). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l⁻¹) and gibberellic acid (GA₃; 0.5 mg l⁻¹). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).     

Micropropagation of commercially cultivated Henna (Lawsonia inermis) using nodal explants

Lawsonia inermis Linn. (Mehandi) is cultivated as cash crop in India particularly in Sojat area of Pali district, Rajasthan. Present investigation describes an efficient regeneration system for elite genotype of L. inermis using nodal segments. Optimum response in terms of percent cultures responding, days to bud break and average shoot length was observed on MS medium supplemented with 6-benzylaminopurine (BA; 2.0 mg l⁻¹). Shoot multiplication was influenced by plant growth regulators, repeated transfer of explants and addition of ammonium sulphate. Maximum shoots were regenerated on MS medium supplemented with BA (0.25 mg l⁻¹), kinetin (Kn; 0.25 mg l⁻¹), indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and ammonium sulphate (150 mg l⁻¹). To reduce resources, time and labours costs, we have also attempted ex vitro rooting of shoots. About 95 % shoots were rooted ex vitro on soilrite after treatment with indole-3-butyric acid (IBA; 300 mg l⁻¹) and 2-naphthoxy acetic acid (NOA; 100 mg l⁻¹) and establishment in soil successfully.Keyword: Ex vitro rooting, Lawsonia inermis, Plant growth regulator, In vitro propagation, Repeated transfer     

An Experimental Study on Citric Acid Production by Aspergillus niger Using Gelidiella acerosa as a Substrate

Citric acid (CA) is the most important commercial product which is produced by using various sugar substrates in the terrestrial environment. The present study made an attempt to produce citric acid by the fungal strain Aspergillus niger from red seaweed Gelidiella acerosa is the best alternative to sugar substrate in the marine environment. In this study three types of production media were prepared including control (sucrose) by following standard fermentation conditions. The acid production was indicated by the reduction of pH levels. The control medium gave the highest yield of 80 g/l at pH 1.5 and the medium containing crude seaweed powder and other compositions gave the yield of 30 g/l at pH 3.5 whereas the medium containing crude seaweed and 10% sucrose gave the yield of 50 g/l at pH 3.0. When calculating the benefit cost ratio, crude seaweed powder and 10% sucrose yielded 50 g of citric acid at the lower cost of Rs. 35, whereas the other two media gave the yield of 80 and 30 g respectively with the cost of Rs. 77 and 28. In economic point of view, the medium containing seaweed and 10% sucrose showed more benefit with lower cost.     

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase,a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN₁ and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers’ fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 μg mL⁻¹) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.