Cadmium biosorption on Spirulina platensis biomass

Dry biomass of Spirulina platensis re-hydrated for 48 h was employed as a biosorbent in tests of cadmium(II) removal from water. Various concentrations of biomass (from 1 to 4 g l⁻¹) and metal (from 100 to 800 mg l⁻¹) were tested. Low biomass levels (X_o  2 g l⁻¹) ensured metal removal up to 98% only at Cd₀= 100 and 200 mg l⁻¹, while X_o  2.0 g l⁻¹ were needed at Cd₀ = 400 mg l⁻¹ to achieve satisfactory results. Whereas X_o = 4.0 g l⁻¹ was effective to remove up to Cd₀ = 500 mg l⁻¹, a further increase in metal concentration (Cd₀ = 600 and 800 mg l⁻¹) led to progressive worsening of the system performance. At a given biomass levels, the kinetics of the process was better at low Cd²⁺ concentrations, while, raising the adsorbent level from 1.0 to 2.0 g l⁻¹ and then to 4.0 g l⁻¹, the rate constant of biosorption increased by about one order of magnitude in both cases and the adsorption capacity of the system progressively decreased from 357 to 149 mg g⁻¹.     

Phenol inhibition of biological nutrient removal in a four-step sequencing batch reactor

Nutrient removal from synthetic wastewater was investigated using a four-step sequencing batch reactor (SBR) at different phenol (C₆H₅OH) concentrations in order to determine the inhibition effects of phenol on biological nutrient removal. The nutrient removal process consisted of anaerobic, oxic, anoxic, and oxic phases with hydraulic residence times (HRT) of 1 h/3 h/1 h/1 h and a settling phase of 3/4 h. Solids retention time (SRT) was kept constant at 10 days in all experiments. Initial phenol concentrations were varied between 0 and 600 mg l⁻¹ at seven different levels. The effects of phenol on COD, NH₄-N, and PO₄-P removals and effluent nutrient levels were investigated. Phenol was almost completely degraded up to 400 mg l⁻¹ phenol concentration resulting in almost negligible inhibition effects on COD, NH₄-N, and PO₄-P removals. Nutrient removals were adversely affected by phenol at concentrations above 400 mg l⁻¹. Above 95% COD, 90% NH₄-N and 65% PO₄-P removal was obtained for phenol concentrations below 400 mg l⁻¹. The sludge volume index (SVI) was almost constant around 45 ml g⁻¹ for phenol concentrations below 400 mg l⁻¹ but increased to 90 ml g⁻¹ at a phenol level of 600 mg l⁻¹.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Aerobic chromate reduction by chromium-resistant bacteria isolated from serpentine soil

A group of 34 chromium-resistant bacteria were isolated from naturally occurring chromium percolated serpentine soil of Andaman (India). These isolates displayed different degrees of chromate reduction under aerobic conditions. One of the 34 isolates identified as Bacillus sphaericus was tolerant to 800 mg l⁻¹ Cr(VI) and reduced >80% Cr(VI) during growth. In Vogel Bonner broth, B. sphaericus cells (10¹⁰ cells ml⁻¹) reduced 62% of 20 mg l⁻¹ of Cr(VI) in 48 h with concomitant discoloring of yellow medium to white one. Reduction of chromate was pronounced by the addition of glucose and yeast extract as electron donors. In the presence of 4.0 g l⁻¹ of glucose, 20 mg l⁻¹ of Cr(VI) was reduced to 2.45 mg l⁻¹ after 96 h of incubation. Optimum pH and temperature for reduction were 6.0 and 25 °C, respectively. Increase in cell density and initial Cr(VI) concentration increased chromate reduction but was inhibited by metal ions like, Ni²⁺, Co²⁺, Cd²⁺ and Pb²⁺. Experiments with cell-free extracts indicated that the soluble fraction of the cell was responsible for aerobic reduction of Cr(VI) by this organism.     

Decolorization of reactive dyes by mixed cultures isolated from textile effluent under anaerobic conditions

In the present study mixed cultures that could grew in the molasses media were isolated from textile dye effluent and its decolorization activity was studied in a batch system under anaerobic conditions, in order to determine the optimal conditions required for the highest decolorization activity. The optimum pH value for decolorization was determined as 8 for all the dyes tested. In the experiment with pH 8 dye decolorizations by mixed cultures were investigated at about 96.2–1031.3 mg l⁻¹ initial dye concentrations. The highest dye removal rates of mixed cultures were 94.9% for Reactive Red RB, 91.0% for Reactive Black B and 63.6% for Remazol Blue at 953.2, 864.9 and 1031.3 mg l⁻¹ initial dye concentrations respectively within 24 h incubation period. When the Reactive Red RB was used, approximately 82–98% total color removal was obtained at between 96.2 and 953.2 mg l⁻¹ initial dye concentrations after 12 h of incubation at 35 °C. These results show that our enriched mixed cultures have the potential to serve as an excellent biomass for the use in reactive dye removal from wastewaters under anaerobic conditions.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Polyamine and methyl jasmonate-influenced enhancement of betalaine production in hairy root cultures of Beta vulgaris grown in a bubble column reactor and studies on efflux of pigments

Hairy root cultures of red beet (Beta vulgaris) were grown in 3 l bubble column reactor for studying growth and pigment production under the influence of polyamines (PA) and elicitor treatment. Earlier studies with shake flask cultures had shown that combined feeding of spermidine (spd) and putrescine (put) (each 0.75 mM) significantly enhanced betalaine productivity in hairy root cultures of red beet. The present study has been focused on betalaine production in 3 l bubble column bioreactor where the growth pattern and betalaine synthesis under the influence of similar levels of polyamines were followed. A combination of spermidine and putrescine fed to the roots each at levels of 0.75 mM efficiently increased growth and pigment production resulting in 1.23-fold higher biomass (39.2 g FW l⁻¹) and 1.27-fold higher betalaine content (32.9 mg g⁻¹ DW) than control. Treatments with various levels of elicitor-methyl jasmonate (MJ), though progressively retarded biomass, at 40 μM level resulted in a significant increase in betalaine content resulting in 36.13 mg g⁻¹ DW which was 1.4-fold higher than the control. Further higher concentrations of methyl jasmonate treatments supported high as well as rapid accumulation of betalaines, the overall betalaine productivity was hampered mainly because of the inhibitory action on biomass. Pigment release studies with cetyl trimethyl ammonium bromide (CTAB) resulted in optimization of concentration for better efflux of betalaines without showing any inhibitory effect on hairy root viability. These studies on product enhancement and on-line extraction of pigment are useful for developing a bioreactor system for betalaine production using B. vulgaris hairy root cultures. In particular the use of elicitors and efflux studies provide an insight for integrating unit operations and developing a process for continuous operation and higher production of phytochemicals.     

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 g N⁻¹ m⁻³ ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mg l⁻¹, the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5 min, being independent of the concentrations, and the lower detection limit was 0.5 mg l⁻¹ BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD₅ method. Accordingly, a good correlation between the sensor and the conventional BOD₅ was obtained (r = 0.989). The system has to evolve the highly sensitive BOD determination.     

Mycelial pellet formation by Penicillium ochrochloron species due to exposure to pyrene

Five indigenous fungal strains with characteristics of the genus Penicillium capable of degrading and utilizing pyrene, as sole carbon source were isolated from soil of a former gas work site. Two strains were identified as Penicillium ochrochloron. One of the strains was able to degrade a maximum of 75% of 50 mg l⁻¹ pyrene at 22 °C during 28 days of incubation. The presence of pyrene in the medium resulted in an aggregation of hyphae into pellets by the two Penicillium ochrochloron strains. Formation of pellets was observed after 48 h of incubation with difference in size and texture between the two strains. This indicated the individual variation within the same genus of fungi. However, remaining strains did not show this behavior even though they were capable of utilizing pyrene as sole carbon source. The macro- and microscopic morphology of fungal pellets was studied using scanning electron microscopy. It was found that the addition of varying concentration of pyrene ranging from 10 to 50 mg l⁻¹ in the medium influenced shape and structure of the mycelial pellets. A two-fold increase in hyphal branching (with concomitant decrease in the average hyphal growth unit) was observed at a concentration of 10 mg l⁻¹. The relevance of fungal growth and morphology for bioremediation of polycyclic aromatic hydrocarbons (PAHs) contaminated sites are discussed.     

Negative effect of discharging vinification lees on soils

In this work, vinification lees from Galicia (Spain) were chemically analysed and compared with the composition of vinification lees from other regions and residues. Moreover, vinification lees were submitted to biological test employing cress, spring barley and ryegrass seeds. The evaluated vinification lees were rich in nutrients that are essential for plants, like P (2520 mg kg⁻¹), K (36,738 mg kg⁻¹) and Mg (462 mg kg⁻¹), but have low pH (3.9) and high C/N ratio. However, when vinification lees were submitted to biological tests, no germination was observed for garden cress and ryegrass seeds and almost no germination for spring barley seeds, showing the negative effect of discharging lees on crop fields.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition

A novel bloom of Alexandrium minutum occurred in an inner basin of the Cape Town harbour from November 2003 to February 2004. Cellular concentrations reached a maximum of 1.4 × 10⁸ cells l⁻¹ during the mid-December period with corresponding chlorophyll a concentrations of 243 mg m⁻³. Primary productivity measurements conducted during the latter part of the bloom revealed a maximum assimilation number of 11.17 mg C mg Chl a⁻¹ h⁻¹ during the middle of the day. Productivity during this post-peak period was sustained largely by the reduced nitrogen species NH₄ and urea (96%) as measured using ¹⁵N tracer techniques. The large subunit ribosomal DNA sequence of A. minutum isolates from Cape Town harbour was identical to conspecifics collected in Western Europe and in Australia. The composition of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was limited to gonyautoxins (GTX1-GTX4). This profile combined with evidence of a low toxin cell quota (1.5 fmol GTX cell⁻¹) supports a close association of this taxon with other members of the A. minutum species complex, particularly from Europe. Toxin analysis from black mussels collected during this bloom indicated that the accumulated PSP toxins originated from A. minutum and not from Alexandrium catenella as is most often the case along the South African coast.     

Purification and immobilization of recombinant variants of Brevundimonas diminuta glutaryl-7-aminocephalosporanic acid acylase expressed in Escherichia coli cells

Cyclotides are naturally occurring mini-proteins that have a diverse range of therapeutically useful biological activities. Although a choice of approaches is available for cyclotides synthesis; most studies have involved the use of peptides extracted from plants. In order to facilitate the screening for structure-activity studies or to exploit them in drug development, a convenient and reliable route for the biosynthesis of cyclotides is of vital importance.

Callus, suspension cultures and hydroponic plants of Oldenlandia affinis were established and have been evaluated for effective cyclotides production processes. The specific accumulation of kalata B1 was affected by cell differentiation as well as agitation; highest accumulation of 2.7 mg g⁻¹ dry weight was detected in agitated hydroponic plant cultures resulting in a productivity of 1.4 mg kalata B1 l⁻¹ day⁻¹.     

Effects of time and dietary iron on tissue iron concentration as an estimate of relative bioavailability of supplemental iron sources for ruminants

Two experiments were conducted to investigate the effects of time and dietary Fe on tissue Fe concentrations following short-term, high level supplementation for use as a bioassay procedure for supplemental Fe sources for ruminants. In Experiment 1, 28 wethers were allotted randomly to four experimental diets which were fed for 15 or 30 days. The basal maize–soyabean meal–cottonseed hulls diet (193 mg kg⁻¹ Fe) was supplemented with 0, 400, 800 or1200 mg kg⁻¹ added Fe from reagent grade ferrous sulfate (FeSO₄·7H₂O). Iron concentrations in liver, kidney, and spleen increased (P<0.05) as dietary Fe increased; however, muscle, heart, and bone Fe concentrations were unaffected. A logarithmic transformation of liver or kidney Fe concentrations at 30 days regressed on added dietary Fe produced the best fits to a linear model. In Experiment 2, bioavailability of Fe from three feed grade ferrous carbonates known to differ (carbonates A, B, and C) was compared to that from reagent grade ferrous sulfate. The dietary treatments fed for 30 days included the above basal diet (90 mg kg⁻¹ Fe) supplemented with 0, 300, 600 or 900 mg kg⁻¹ added Fe from ferrous sulfate or 600 mg kg⁻¹ Fe from ferrous carbonates A, B, or C. Liver Fe concentrations from sheep fed ferrous sulfate were numerically greater than those of animals fed the carbonate sources or control diet. Kidney Fe concentrations from lambs fed ferrous sulfate at 600 mg kg⁻¹ Fe or carbonate-A were greater (P<0.05) than those fed carbonates B or C. Iron concentrations in spleen were lower (P<0.05) in lambs fed carbonate-B than for those fed 600 mg kg⁻¹ Fe as ferrous sulfate, but were similar to other carbonates. Overall average bioavailability estimates based on multiple regression slope ratios for the three tissues were ferrous sulfate 1.00, carbonate-A 0.55, carbonate-B 0.00, and carbonate-C 0.20. Estimates for carbonates A and C were similar to those based on hemoglobin concentrations reported previously for young swine supplemented at dietary concentrations near the requirement.     

The adsorption of copper(II) ions on to dehydrated wheat bran (DWB): determination of the equilibrium and thermodynamic parameters

The adsorption of copper(II) ions on to dehydrated wheat bran (DWB), a by-product of the flour process, was investigated as a function of initial pH, temperature, initial metal ion concentration and adsorbent dosage. The optimum adsorption conditions were initial pH 5.0, initial copper concentration 100 mg l⁻¹, temperature 60 °C and adsorbent dosage 0.1 g. The adsorption equilibrium was described well by the Langmuir isotherm model with maximum adsorption capacity of 51.5 mg g⁻¹ of copper(II) ions on DWB. The observation of an increase in adsorption with increasing temperature leads to the result that the adsorption of copper(II) ions on DWB is endothermic in nature. The thermodynamic parameters such as enthalpy, free energy and entropy changes were calculated and these values show that the copper(II)-DWB adsorption process was favoured at high temperatures.     

Medium optimization for the production of the secondary metabolite squalestatin S1 by a Phoma sp. combining orthogonal design and response surface methodology

In the present work, a combined statistical approach of orthogonal design (L₂₇(3¹³)), response surface techniques and polynomial regression were applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a fungus (a Phoma species). Optimal conditions for maximal titres and productivity were determined based on 13 parameters at three different levels. Initially, a screening design methodology was used to evaluate the process variables, which were relevant to S1 titre and the response surfaces applied to find optimal regions for production. The sources of carbon and concentration, and their interactions with oily precursors were statistically significant factors. The combined orthogonal design and response surface methodology predicted optimal conditions for of 273 mg l⁻¹ of squalestatin S1. Confirmatory experiments of the optimal medium composition produced titres of 434 mg l⁻¹ in a 5-day fermentation at 25 °C. This represented a 60% improvement in the maximum titre predicted, and a two-fold higher productivity when compared with reported S1 yields of various fungal species. This combined statistical approach enables rapid identification and integration of key medium parameters for optimising secondary metabolite production and could be very useful in pharmaceutical screening programmes.     

Effect of iron concentration on hydrogen fermentation

The effect of the iron concentration in the external environment on hydrogen production was studied using sucrose solution and the mixed microorganisms from a soybean-meal silo. The iron concentration ranged from 0 to 4000 mgFeCl₂ l⁻¹. The temperature was maintained at 37°C. The maximum specific hydrogen production rate was found to be 24.0 mlg⁻¹ VSSh⁻¹ at 4000 mgFeCl₂ l⁻¹. The specific production rate of butyrate increased with increasing iron concentration from 0 to 20 mgFeCl₂ l⁻¹, and decreased with increasing iron concentration from 20 to 4000 mgFeCl₂ l⁻¹. The maximum specific production rates of ethanol (682 mgg⁻¹ VSSh⁻¹) and butanol (47.0 mgg⁻¹ VSSh⁻¹) were obtained at iron concentrations of 5 and 3 mgFeCl₂ l⁻¹, respectively. The maximum hydrogen production yield of 131.9 mlg⁻¹ sucrose was obtained at the iron concentration of 800 mgFeCl₂ l⁻¹. The maximum yields of acetate (389.3 mgg⁻¹ sucrose), propionate (37.8 mgg⁻¹ sucrose), and butyrate (196.5 mg g⁻¹ sucros) were obtained at iron concentrations of 3, 200 and 200 mgFeCl₂ l⁻¹, respectively. The sucrose degradation efficiencies were close to 1.0 when iron concentrations were between 200 and 800 mgFeCl₂ l⁻¹. The maximum biomass production yield was 0.283 gVSSg⁻¹ sucrose at an iron concentration of 3000 mgFeCl₂ l⁻¹.     

Oxidation of hydrogen sulphide in sour gas by Chlorobium limicola

The aim of this work was to assess the potential for bacterial oxidation of hydrogen sulphide as a purification method of sour gas. Using a continuous culture of Chlorobium limicola, high efficiencies of oxidation of both soluble and gaseous sulphide were achieved, with efficiencies for the latter exceeding 95%. Sulphide added as aqueous sodium sulphide was converted to sulphur and sulphate with almost total removal of the initial 100 mg S l⁻¹ within 24 h. Gaseous sulphide was oxidized at an efficiency of 95% (approximately 3 mmol S h⁻¹ (unit biomass Abs)⁻¹) over 1 h runs at a gas flow rate of 60 ml min⁻¹. With a sulphur recovery system to prevent sulphur accumulation, an efficiency of 70% was maintained. Biological removal of sulphide represents a potentially important biotechnological process, with high potential for viable scale up.     

Determination of digoxin in serum samples using a flow-through fluorosensor based on a molecularly imprinted polymer

This work describes the development of a competitive flow-through FIA assay for digoxin using a molecularly imprinted polymer (MIP) as the recognition phase. In previous work, a number of non-covalent imprinted polymers were synthesised by “bulk” polymerisation. The digoxin binding and elution characteristics of these MIPs were then evaluated to obtain a highly selective material for integration into a sensor. The optimum MIP was synthesised by photo-initiated polymerisation of a mixture containing digoxin, MAA, EDGMA and AIBN in acetonitrile. The bulk polymer was ground and sieved and the template removed by Soxhlet extraction in MeOH/ACN. The MIP was packed into a flow cell and placed in a spectrofluorimeter to integrate the reaction and detection systems. The physical and chemical variables involved in digoxin determination by the sensor (nature and concentration of solution, flow rates, etc.) were optimised. Binding with the non-imprinted polymer (NIP) was also analysed. The new fluorosensor showed high selectivity and sensitivity, a detection limit of 1.7 × 10⁻² μg l⁻¹, and high reproducibility (R.S.D. of 1.03% and 1.77% for concentrations of 1.0 × 10⁻³ and 4.0 × 10⁻³ mg l⁻¹, respectively). Selectivity was tested by determining the cross-reactivity of several compounds with structures analogous to digoxin. Under the assay conditions used, in which the potential interfering compounds were in concentrations 100 times higher than that of the analyte, no interference was recorded. The proposed fluorosensor was successfully used to determine digoxin concentration of human serum samples.