Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii

A laboratory study was conducted on the removal of nitrogen and phosphorus from piggery wastewater during growth of Botryococcus braunii UTEX 572, together with measurements of hydrocarbon formation by the alga. The influence was tested of the initial nitrogen and phosphorus concentration on the optimum concentration range for a culture in secondarily treated piggery wastewater. A high cell density (> 7 g L^–1 d. wt) was obtained with 510 mg L^–1 NO₃-N. Growth increased with nitrogen concentration at the basal phosphorus concentration (14 mg P L^–1). The growth rate was nearly independent ( = 0.027 0.030 h^–1) of the initial phosphate concentration, except under conditions of phosphate deficiency ( = 0.019 h^–1). B. braunii grew well in piggery wastewater pretreated by a membrane bioreactor (MBR) with acidogenic fermentation. A dry cell weight of 8.5 mgL^–1 and hydrocarbon level of 0.95 gL^–1 were obtained, and nitrate was removed at a rate of 620 mg NL^–1. These results indicate that pretreated piggery wastewater provides a good culture medium for the growth and hydrocarbon production by B. braunii.     

Dibenzothiophene desulfurization in hydrocarbon environment by Staphylococcus sp. resting cells

Staphylococcus sp. strain S3/C desulfurized dibenzothiophene/n-hexadecane (3 mg ml^–1) in a hydrocarbon aqueous biphasic culture. The resting cells decreased the sulfur content of the hydrocarbon phase by 57% at 2.2 mg l^–1 h^–1 in the absence of any additional carbon and sulfur source.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

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.     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Mixotrophic growth ofChlorella sorokiniana in outdoor enclosed photobioreactor

Chlorella sorokiniana was cultured in heterotrophic or mixotrophic mode in outdoor enclosed tubular photobioreactor. The culture temperature was maintained at 32–35 °C. At night, theChlorella culture grew heterotrophically, and 0.1 M glucose was completely consumed. The biomass growth yield of glucose was 0.35 ± 0.001 g-biomass g-glucose^–1. During the day, the algal culture grew mixotrophically and the biomass growth yield was 0.49 g-biomass g-glucose^–1 in low density culture (initial biomass concentration, X_o = 2 g l^–1), 0.56 g-biomass g-glucose^–1 in medium density culture (X_o = 4 g l^–1) and 0.46 g-biomass g-glucose^–1 in high density culture (X_o = 7 g l^–1). The daily area productivity of the culture, with X_o = 4 g l^–1 corresponded to 127 g-biomass m^–2 d^–1 during the day and 79 g-biomass m^–2 d^–1 during the night. In all the cultures, the dissolved O₂ concentration increased in the morning, reached the maximum value at noon, and then decreased in the afternoon. The dissolved CO₂ concentration remained at 3 mBar in the morning and increased in the afternoon. Glycolate was not found to accumulate in culture medium.     

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l^–1 to a chemically defined medium containing 140 g glucose l^–1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l^–1 to 47.7 g l^–1. With 40 g NaCl l^–1 at a dilution rate of 0.065 h^–1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.     

Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans

Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4 g l^–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5 g l^–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16 g l^–1 h^–1 were obtained when the C/N ratios in the feed were 10, 20 and 100 g octanoic acid g^–1 ammonium nitrate, respectively.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Plant regeneration from seedling explants of perennial Glycine species

More than 5000 cultures, from 30 accessions of six Glycine species, were established to assess the rôle of plant genotype in the response to an agar-solidified culture medium containing B5 salts and vitamins, 3% w/v sucrose, 1.1 mg 1^–1 BAP and 0.005 mg 1^–1 IBA, already known to induce shoot regeneration in callus of G. clandestina. Shoot initiation was obtained in a variety of explants from G. canescens, G. falcata, G. latrobeana and G. tomentella. With the exception of G. latrobeana, development of buds into shoots followed transfer to B5-based medium with 0.2 mg^–1 BAP and 0.005 mg 1^–1 IBA. Shoots readily produced roots in hormone-free half-strength B5 medium. In G. latrobeana, both extension and rooting occurred on this medium. Shoot regeneration was obtained in 12 of 30 accessions evaluated, but one accession of G. canescens, G1171, produced shoots and plantlets at a consistently higher frequency than other accessions, with plantlet recovery in more than 70% of the cultures. Bud formation in callus of G. canescens G1171 also occurred if BAP was replaced by 1.0 mg 1^–1 kinetin, 2i-p or zeatin, albeit at a lower frequency.     

Enhancement of phase separation by the addition of de-emulsifiers to three-phase (diesel oil/biocatalyst/aqueous phase) emulsion in diesel biodesulfurization

Ethanol, added as a de-emulsifier to separate oil and biocatalyst (or bacterial cells) from a three-phase (oil/biocatalyst/aqueous phase) emulsion, formed in diesel biodesulfurization employing Gordonia nitida, improved oil recovery by centrifugation from about 50% in its absence to almost 100% at 3% (v/v). The biocatalyst recovered with ethanol addition showed similar specific growth rates (0.03 h^–1) and dibenzothiophene desulfurization rates (6–7.2 mol l^–1 h^–1) to those (0.03 h^–1 and 7.1 mol l^–1, respectively) of the biocatalyst recovered with no ethanol addition. The desulfurization activity significantly increased as the number of the repeated recovery and reuse of the biocatalyst.     

Production potential of eicosapentaenoic acid by the diatom Nitzschia laevis

To investigate the production potential of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, the growth characteristics and fatty acid composition of the cells were studied under photoautotrophic, mixotrophic and heterotrophic conditions of growth. The specific growth rate and maximum biomass concentration were respectively 0.466 d^–1 and 2.27 g l^–1 for mixotrophic culture, 0.344 d^–1 and 2.04 g l^–1 for heterotrophic culture, and 0.167 d^–1 and 0.5 g l^–1 for photoautotrophic culture, respectively. As for EPA production, the yield and productivity were respectively 52.32 mg l^–1 and 10.46 mg l^–1 d for mixotrophic culture, 35.08 mg l^–1 and 6.37 mg l^–1 d for heterotrophic culture, and 6.78 mg l^–1 and 3.39 mg l^–1 d for photoautotrophic culture, respectively. Results suggest that mixotrophic culture is the most suitable growth mode for the production of EPA by the diatom Nitzschia laevis. The results are useful for the development of a cost-effective fermentation process for EPA production by Nitzschia laevis.     

Plant Regeneration from Mesophyll Protoplasts of Echinacea Purpurea

An efficient plant regeneration system was developed from isolated protoplasts of Echinacea purpurea L. using an alginate block/liquid culture system. Viable protoplasts could be routinely isolated from young leaves of Echinacea seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase and 0.3 mol l^–1 mannitol. Purified protoplasts were embedded in 0.6% Na-alginate block at a density of 1 × 10⁵/ml and cultured in a modified MS medium containing 0.3 mol l^–1 sucrose, 2.5 µmol l^–1 BA and 5.0 µmol l^–1 2,4-D. Cell colonies were observed after 4 weeks of culture, and the protoplast-derived colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS medium supplemented with 1.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Shoot organogenesis from protoplast-derived callus was induced on MS medium supplemented with 5.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Complete plantlets were obtained from the regenerated shoots on MS basal medium. The protoplast to plant regeneration protocol developed in this study provides the prerequisite for creating novel genotypes of this valuable medicinal species through genetic manipulation.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 g L^–1 day^–1 after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 g L^–1 day^–1, a productivity of 5 g L^–1 day^–1 was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.     

Fed-batch fermentation of Lactobacillus lactis for hyper-production of l-lactic acid

A fed-batch fermentation of Lactobacillus lactis to produce l-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g l-lactic acid l^–1 (97% yield) was obtained. The maximal dry cell was 2.7 g l^–1 and the average l-lactic acid productivity was 2.2 g l^–1 h^–1.     

Intensive rotifer cultures using chemostats

Continuous production of the rotifer Brachionus plicatilis rotundiformis (S-type) in an intensive chemostat culture system has been investigated. The production dynamics of rotifers in relation to different flow rates and feed regimes show that the growth rate and production depends on the type of algal feed and flow rate utilized in the culture system. It was possible to achieve a mean production of up to 318.84 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 6 1 h^–1 in 100 1 chemostats and up to 261.21 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 40 1 h –1 while using 1 m³ capacity rotifer chemostats as production units. The 3 fatty acid composition of rotifers while using Chlorella and Nannochloropsis in the culture system has been described. The results of this investigation show that the rotifer productivity in the continuous culture system is considerably higher than in any of the conventional culture systems described to date for aquacultural purposes.This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS), Kuwait, under a contract research project code 86-04-02.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.