Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

A chemically-defined medium for production of compactin by Penicillium citrinum

A mutant strain of Penicillium citrinum grown in a chemically-defined production medium, yielded 145 mg compactin l^–1. The medium also facilitated spectrophotometric analysis of compactin. Addition of KH₂PO₄in the production medium did not increase the compactin production, while addition of a surfactant, Tween 80, increased compactin to 175 mg l^–1. Inoculation with 10⁷ spores ml^–1 and initial pH of 6.5–7 were the most suitable for compactin production.     

Two extracellular endoxylanases from alkaliphilic Bacillus firmus differ in their synthesis

To investigate the synthesis of two extracellular endoxylanases, xylan-binding and unbound xylanases from an alkaliphilic Bacillus firmus, washed cells were incubated in alkaline mineral salt media containing various carbon sources. The 23 kDa xylan-binding endoxylanase (XBE), which hydrolyses insoluble xylan, was produced before the 45 kDa, unbound endoxylanase. All the carbon sources tested at 5 mg ml^–1, including glucose, induced production of XBE but the unbound xylanase was totally repressed by glucose. The production of XBE increased when glucose concentration increased but was not synthesized until the glucose in the medium was less than 1 mg ml^–1.     

Hydrogen accumulation by H2-uptake negative strains of Rhizobium

Summary Hydrogen evolution from root nodules has been reported to decrease the efficiency of the nitrogen fixing system. Mutants ofRhizobium meliloti andRhizobium leguminosarum were selected which were deficient in H₂-uptake capacity (Hup^–). The relative efficiency of the nitrogen fixation for both species assessed with C₂H₂ reduction was 0.66.The hydrogen production was monitored using a simple root incubation method. As such, hydrogen production up to 3.83 and 15.57 ml.day^–1.g^–1 plant dry weight were recorded forPisum sativum — Rhizobium leguminosarum 4.20 Hup^– andMedicago sativa — Rhizobium meliloti 1.5 Hup^– respectively. In a closed container (250 ml), hydrogen concentrations up to 20% (v/v) could be reached in the root phase ofMedicago sativa in a time period of 320 hours.     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

Growth promotion of Prunus rootstocks by root treatment with specific bacterial strains

A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach–almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6–3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the non-treated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log₁₀ CFU ml^–1 (ca 10 log₁₀ CFU L^–1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log₁₀ CFU g f.w.^–1 root material at early stages of development and decreased to 4 log₁₀ CFU g f.w.^–1 after several months of development. The best plant growth-promoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.     

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l^–1 were 20 g dry cell wt l^–1, 1.9 FPU ml^–1 and 4.8 FPU mg^–1 protein respectively, while on 40 g glucose l^–1 were 25 g dry cell wt l^–1, 4.5 FPU ml^–1 and 6.2 FPU mg^–1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg^–1 protein) than was produced by other T. reesei mutants (3.6 FPU mg^–1 protein).     

Nitrogen fixation by Nodularia spumigena Mertens (Cyanobacteriaceae). 1: Field studies and the contribution of blooms to the nitrogen budget of the Peel-Harvey Estuary,Western Australia

Variations in nitrogen fixation (acetylene reduction) by Nodularia spumigena blooms in the Peel-Harvey estuarine system were examined with respect to spatial (sampling station location, and depth) and temporal (seasonal and diurnal) distribution. The annual contributions of nitrogen fixation by the blooms to the nitrogen budget of the estuary were estimated to range from 309 to 713t. Contributions by nitrogen fixation were similar to the riverine inputs in the Harvey Estuary, but lower in the Peel Inlet.The Harvey Estuary had higher biomass and total fixation rates (to 0.4 nmol C₂H₂ · ml^–1 h^–1), but the heterocyst nitrogen fixation rates were greater in the Peel Inlet (to 9 × 10^–1 nmol C₂H₂ · heterocyst^–1 · h^–1). Nitrogen fixation decreased with depth in response to light, though other factors also appeared to be involved. The rates of fixation decreased concurrently with increasing bloom age, total soluble inorganic nitrogen and salinities. Maximum daily fixation rates occurred in the early morning.     

Production of S-adenosyl- L-methionine by a mutant strain of Kluyveromyces lactis

S-Adenosyl-l-methionine (AdoMet) was produced by a mutant strain Kluyveromyces lactis AM-65 grown on whey. A full factorial design method of three factors – (NH₄)₂SO₄ (factor x ₁), corn steep liquor (factor x ₂) and l-methionine (factor x ₃) on three levels – was used to determine the optimal medium conditions for the production of AdoMet. A time course shake-flask experiment in optimal whey medium (x ₁=3.1 g l^–1, x ₂=12.7 g l^–1, x ₃=4.6 g l^–1) was also carried out and the results confirmed the results of the factorial design and subsequent quadratic modelling and optimization of AdoMet production which reached 90 mg g^–1 cell dry wt.     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Extractive cultivation of Lactococcus lactis using a polyethylene glycol/MgSO4 · 7H2O aqueous two-phase system to produce nisin

To relieve lactic acid inhibition, an aqueous two-phase system (ATPS) was used to grow Lactococcus lactis. Its composition was 11% (w/v) PEG 20000/3.5% (w/v) MgSO₄ 7H₂O. In this ATPS medium, the cells were completely partitioned in the bottom phase, and lactic acid had the biggest partition coefficient of the eight ATPS media tested. The cell biomass in this medium was 0.64 mg ml^–1, only 60% of that of the control medium, but nisin production (803 IU ml^–1) was enhanced by 33%. The increase in nisin was explained as a result of extraction of lactic acid from the bottom phase to the top one. The changes of tie-line length and phase volume ratio for the identical tie line could affect cell growth and nisin accumulation.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

Control of xylanase production without protease activity in Bacillus sp. by selection of nitrogen source

Maximum xylanase activity, of 380 IU ml^–1, with negligible protease activity, occurred when Bacillus SSP-34 was grown for 96 h with yeast extract and peptone each at 0.25%. Other concentrations of the combination gave xylanase activities less than 66% of that with the optimum nitrogen source concentration and protease activities in the range of 0.01–0.045 IU ml^–1.     

Respiratory stimulus in Rhizobium sp. by legume lectins

High molecular weight lectins (> 100 kDa) from seeds of the legumes Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Phaseolus vulgaris (PHA) and Vatairea macrocarpa (VML), temporarily stimulate the respiration of Rhizobium tropici-CIAT899 and R. etli-CFN42. These stimulants were significant (P < 0.05) in bacterial suspensions (> 2.85 mg dry biomass ml^–1), having at least 6200 molecules of lectins per bacteria. The VML (20 g ml^–1), induced specific O₂ demand of 2.3–2.5 M O₂ min^–1 mg dry biomass^–1, in CFN42 and CIAT899, respectively. However, CnBr, CFL and PHA induced smaller demands of O₂ (5×), in both strains. The order of affinities of the lectins was approximately VML > PHA > CFL > CnBr, with regard to respiratory stimuli in CIAT899 strain. The co-administration of 10 g VML ml^–1 and 9.8 M galactose, in CIAT899 suspensions, reduced the respiratory stimuli significantly in relation to the treatment with VML alone. These respiratory stimuli, induced by the lectins, increase the significance of the interaction lectin × Rhizobium in terms of bacterial physiology. Its understanding could be important in relation to bacterial symbiotic behaviour.