João Carlos Monteiro de Carvalho

Effect of reducing the light intensity on the growth and production of chlorophyll by Spirulina platensis

Abstract The influence of light intensity reduction on Spirulina platensis cultivation was investigated, using urea and KNO 3 as nitrogen sources. The reduction of light intensity from 5 to 2 klux was studied both on the 9th and the 13th day of cultivation. Increases of up to 29% in the total chlorophyll production were observed for the cultivations with light intensity reduction, in comparison with the cultivations carried out at fixed light intensities.

CULTIVATION OF ARTHROSPIRA (SPIRULINA) PLATENSIS (CYANOPHYCEAE) BY FED-BATCH ADDITION OF AMMONIUM CHLORIDE AT EXPONENTIALLY INCREASING FEEDING RATES†

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was cultivated under light‐limited conditions in 5‐L open tanks by daily supplying NH4Cl as nitrogen source. Exponentially increasing feeding rates were adopted to prevent ammonia toxicity. The total feeding time (T) was varied between 12 and 20 days, and the starting (m0) and total (mT) quantities of the nitrogen source per unit reactor volume were varied in the ranges 0.19–1.7 mM and 2.3–23.1 mM, respectively. This intermittent addition of the nitrogen source prevented ammonia from reaching inhibitory levels and ensured final cell concentrations (Xm) and cell productivities (Px) comparable with those of batch runs with KNO3. Moreover, the lower nitrogen addition due to the use of NH4Cl rather than KNO3 allowed for higher nitrogen‐to‐cell conversions (Yx/n). These results were evaluated using three‐factor, five‐level, central composite experimental planning, combined with the response surface methodology, selecting T, m0, and mT as the independent variables and Xm, Px, and Yx/n as the response variables. This approach allowed us to identify, through the simultaneous optimization of the variables, T=16 days, m0=1.7 mM, and mT=21.5 mM as the best conditions for A. platensis cultivation at 72 μmol photons·m−2·s−1. Under these conditions, a maximum cell concentration of 1239 mg ·L−1 was obtained, which is a value comparable with that obtained using KNO3 as nitrogen source and nearly coincident with the theoretical one estimated by the response surface methodology.

Fed-batch cultivation of Arthrospira (Spirulina) platensis: potassium nitrate and ammonium chloride as simultaneous nitrogen sources.

Arthrospiraplatensis was cultivated in minitanks at 13 klux, using a mixture of KNO(3) and NH(4)Cl as nitrogen source. Fed-batch daily supply of NH(4)Cl at exponentially-increasing feeding rate allowed preventing ammonia toxicity and nitrogen deficiency, providing high maximum cell concentration (X(m)) and high-quality biomass (21.85 mg chlorophyll g cells(-1); 20.5% lipids; 49.8% proteins). A central composite design combined to response surface methodology was utilized to determine the relationships between responses (X(m), cell productivity and nitrogen-to-cell conversion factor) and independent variables (KNO(3) and NH(4)Cl concentrations). Under optimum conditions (15.5mM KNO(3); 14.1mM NH(4)Cl), X(m) was 4327 mg L(-1), a value almost coincident with that obtained with only 25.4mM KNO(3), but more than twice that obtained with 21.5mM NH(4)Cl. A 30%-reduction of culture medium cost can be estimated when compared to KNO(3)-batch runs, thus behaving as a cheap alternative for the commercial production of this cyanobacterium.

Metal biosorption onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris: multi-metal systems.

Binary and ternary systems of Ni(2+), Zn(2+), and Pb(2+) were investigated at initial metal concentrations of 0.5, 1.0 and 2.0mM as competitive adsorbates using Arthrospira platensis and Chlorella vulgaris as biosorbents. The experimental results were evaluated in terms of equilibrium sorption capacity and metal removal efficiency and fitted to the multi-component Langmuir and Freundlich isotherms. The pseudo second order model of Ho and McKay described well the adsorption kinetics, and the FT-IR spectroscopy confirmed metal binding to both biomasses. Ni(2+) and Zn(2+) interference on Pb(2+) sorption was lower than the contrary, likely due to biosorbent preference to Pb. In general, the higher the total initial metal concentration, the lower the adsorption capacity. The results of this study demonstrated that dry biomass of C. vulgaris behaved as better biosorbent than A. platensis and suggest its use as an effective alternative sorbent for metal removal from wastewater.

LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (KGFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REMLPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch process: optimization by response surface methodology.

We studied the effect of reactor filling time (T) (3–5 h), initial mass of inoculum (M) (1000–2100 g), and exponential time decay constant for the substrate feed rate (K) (0.6–1.6 h−1) on ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch culture. The highest ethanol productivity (16.9 g/[L·h]) occurred at T=3h, K=1.6 h−1, and M=1300 g. In addition, productivity was affected by both M (for T=3 and 4 h) and K (for T=3 h) and varied inversely with T under any value fixed for M and K. By the quadratic regression multivariable analysis method, equations were determined to estimate ethanol yield and productivity as function of the variables studied (T, K, and M).

Influence of ammonium sulphate feeding time on fed-batch Arthrospira (Spirulina) platensis cultivation and biomass composition with and without pH control

Previous work demonstrated that a mixture of NH(4)Cl and KNO(3) as nitrogen source was beneficial to fed-batch Arthrospira (Spirulina) platensis cultivation, in terms of either lower costs or higher cell concentration. On the basis of those results, this study focused on the use of a cheaper nitrogen source mixture, namely (NH(4))(2)SO(4) plus NaNO(3), varying the ammonium feeding time (T=7-15 days), either controlling the pH by CO(2) addition or not. A. platensis was cultivated in mini-tanks at 30°C, 156 μmol photons m(-2) s(-1), and starting cell concentration of 400 mg L(-1), on a modified Schlösser medium. T=13 days under pH control were selected as optimum conditions, ensuring the best results in terms of biomass production (maximum cell concentration of 2911 mg L(-1), cell productivity of 179 mg L(-1)d(-1) and specific growth rate of 0.77 d(-1)) and satisfactory protein and lipid contents (around 30% each).

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I=108 μmol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m)=5772±113 mg L(-1)) and productivity (P(XS)=1319±25 mg L(-1) d(-1)) at the lowest (D=0.1 day(-1)) and the highest (D=0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudo-steady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables.

A new approach to ammonium sulphate feeding for fed-batch Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor

Arthrospira platensis was cultivated in tubular photobioreactor using different photosynthetic photon flux densities (PPFD) and protocols of (NH4)2SO4 fed‐batch supply. Results were evaluated by variance analysis selecting maximum cell concentration (Xm), cell productivity (Px), nitrogen‐to‐cell conversion factor (YX/N) and biomass, protein and lipid contents as responses. At PPFD of 120 and 240 μmol‐photons/m2 s, a parabolic profile of (NH4)2SO4 addition aiming at producing biomass with 7% nitrogen content ensured Xm values (14.1 and 12.2 g/L, respectively) comparable to those obtained with NaNO3. At PPFD of 240 μmol‐photons/m2 s, Px (1.69 g/Ld) was 36% higher, although the photosynthetic efficiency (3.0%) was less than one‐half that at PPFD of 120 μmol‐photons/m2 s. Biomass was shown to be constituted by about 35% proteins and 10% lipids, without any dependence on PPFD or kind of nitrogen source. These results highlight the possible use of (NH4)2SO4 as alternative, cheap nitrogen source for A. platensis cultivation in tubular photobioreactors.

Effect of medium composition on the production of tetanus toxin by Clostridium tetani.

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N‐Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five‐level star‐shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0 = 9.7 g/L and NZ0 = 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0 = 8.0 g/L and NZ0 = 25.0 g/L).