Swirling flow implementation in a photobioreactor for batch and continuous cultures of Porphyridium cruentum

Light is the main limiting factor in photoautotrophic-intensive production of microorganisms, and improvement of its use is an important concern for photobioreactor design and operation. Swirling flows, which are known to improve mass and photon transfers, were applied to annular light chambers of a photobioreactor and studied by simulation and microalgal culture. Two hydrodynamic conditions were compared: axial flow generating poor radial mixing, and tangential flow generating three-dimensional swirling motion. Batch and continuous cultures of the Rhodophyte Porphyridium cruentum were performed in a 100-L, 1.5-m(2), fully controlled photobioreactor with eight light chambers. The inlet design of these chambers was modified to create the hydrodynamic conditions for comparison. Various intensities of swirling motion were used, characterized by the velocity factor (VF), defined as the ratio between annular chamber flow and inlet aperture sections. Experiments were performed within the range of photon flux densities (PFD) optimizing the yield of light energy transformation into living substance for the species and the temperature used. Culture kinetics with swirling flows generated by apertures of VF = 2, 4, and 9 were compared with pseudoaxial VF = 2 chosen as reference. Batch cultures with VF = 4 swirling flow showed no significant difference, whereas continuous cultures proved more discriminating. Although no significant difference was obtained for VF = 2, a 7% increase of steady-state productivity and a 26% decrease in time required to reach this steady state were obtained with VF = 4 swirling flow. This beneficial effect of swirling flow could have accounted for increased mixing. Conversely, VF = 9 swirling flow resulted in a 9% decrease of steady-state productivity and a 9% increase in the time required to reach this steady state, a negative effect that could have accounted for increased shear stress. CO(2) bioconversion yield at steady state showed a 34% increase for VF = 4. These results suggest that swirling motion makes microalgal cultures more efficient, provided that the resulting adverse effects remain acceptable. Experimental investigation was completed by a theoretical approach in which simulation of continuous cultures of P. cruentum was based on the hydrodynamic conditions achieved in the photobioreactor. Although the results obtained with pseudoaxial flow were correctly predicted, simulations with swirling flow showed a marked enhancement of productivity not observed experimentally. The influence of side effects induced by increased mixing (particularly hydrodynamic shear stress) was considered with respect to modeling assumptions. Comparison of experimental results with theoretical simulation provided a better understanding of the mixing effect, a key factor in improving the efficiency of such bioprocesses.     

平板式光生物反应器中紫球藻培养条件的优化

研究了平板式光生物反应器中紫球藻(Porphyridium cruentumNaegeli)的培养条件,运用均匀设计法对光照强度、通气速率、装液量、接种密度以及pH等影响紫球藻生长的因素进行优化,获得了在平板式光生物反应器中培养紫球藻的最佳条件:光照强度10 000 lx、通气速率350 L.h-1、装液量6 L、藻细胞接种密度1.1×106mL-1、pH9.0。在最佳条件下藻体的生物量产率和生物量产量分别达到0.431 g.L-1.d-1和3.240 g.L-1,最大生长速率达0.652 g.L-1.d-1,胞外多糖含量高达0.665 g.L-1。另外,在培养过程中隔天补充培养液有利于紫球藻生物量的增加和胞外多糖的产生。     

The kinetics of photoinhibition and its recovery in the red alga Porphyridium cruentum

When Porphyridium cruentum cells were illuminated with high fluence rate between 1900 and 4800 mol photons m^-2s^-1, a decrease in the photosynthetic activity of the cells was observed. Within the time frame of 20 min, and under the fluence rates studied, the sum of photons to be absorbed by cells (mg of chlorophyll (Chl), sufficient to initiate photoinhibition was calculated to be 9235.8 mol. The minimal specific light absorption rate to initiate photoinhibition in P. cruentum ranges between 2.29 and 4.26 mol photons s^-1 mg^-1 chl.a. There was a linear relationship between the specific rate of photoinhibition and the specific light absorption rate. A photon number of 2.56×10⁴ mol mg^-1 chl.a photoinhibited photosynthesis instantaneously. At 15°C, no photoinhibitory effect was observed at 2300 mol photons m^-2 s^-1 even after 45 min of illumination. At the other extreme of 35°C, 84% inhibition of photosynthetic activity was observed within 10 min of exposure to 2300 mol photons m^-2 s^-1. Between 20 and 30°C, the photoinhibitory effect was comparable. Photoinhibited P. cruentum cells recovered readily when transferred to low light (90 mol photons m^-2 s^-1) and darkness, and the specific rate of recovery was independent of the light intensity to which the cells were exposed, during the photoinhibitory treatment.Abbreviations Chlorophyll QL, specific light absorption rate Publication No. 28 of the Microalgal Biotechnology Laboratory     

Eicosapentaenoic and arachidonic acids purification from the red microalga Porphyridium cruentum

The polyunsaturated fatty acids (PUFA) eicosapentaenoic and arachidonic acids (EPA and AA), which have several pharmaceutical properties, have been purified from the red microalga Porphyridium cruentum. The process consists of only four main steps: (i) simultaneous extraction and saponification of the microalgal biomass; (ii) urea inclusion method (iii) PUFA esterification (iv) argentated silica gel column chromatography of the urea concentrate. Total AA and EPA recoveries reached 39.5% and 50.8% respectively for a purity 97% for both fatty acids. Therefore, recovery of highly pure PUFA could be improved in organisms that are rich in two or more fatty acids of interest. The results of several procedures for AA and EPA recovery from several authors by using this microalga were compared.     

紫球藻及其胞外多糖对小鼠免疫功能的调节作用

探讨紫球藻及其胞外多糖对免疫低下小鼠免疫功能的调节作用,用环磷酰胺(CY)建立昆明种小鼠免疫功能低下的实验模型。小鼠随机均分6组,其中2组每天分别给予紫球藻藻粉1200、600 mg/kg灌胃,2组给予紫球藻胞外多糖300、150 mg/kg灌胃,正常对照组及CY组用等容积生理盐水灌胃,连续14 d。实验结果表明紫球藻及其胞外多糖均可显著提高免疫抑制小鼠的脾指数、胸腺指数、碳廓清能力、单核细胞吞噬功能,并可对抗环磷酰胺引起的外周血白细胞数目下降,因此紫球藻藻粉及其胞外多糖对小鼠免疫功能具有一定的正向调节作用。毒性实验表明,小鼠腹腔注射300、150 mg/kg紫球藻胞外多糖溶液和分别给予紫球藻藻粉1200、600 mg/kg灌胃,小鼠未出现死亡,也未有明显毒性反应出现的一些指标变化,说明紫球藻藻粉及胞外多糖的安全性较高。     

Glucose uptake of Cytophaga johnsonae studied in batch and chemostat culture

The influence of different physiological states on the glucose uptake and mineralization by Cytophaga johnsonae, a freshwater isolate, was examined in batch and chemostat cultures. At different growth rates under glucose limitation in chemostat cultures, different uptake patterns for ¹⁴C labeled glucose were observed. In batch culture and at high growth rates the glucose uptake potential showed a higher maximum velocity and a much lower substrate affinity than at lower growth rates. These findings and the results of short-term labeling patterns could be explained by two different glucose uptake mechanisms which enable the strain to grow efficiently both at high and low substrate concentrations. Substrate specificity studies showed that a structural change of the C-2 atom of the glucose molecule was tolerated by both systems. The consequences of these results for the ecophysiological classification of the Cytophaga group and for the operation of continuous cultures are discussed.     

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s^–1 damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s^–1. Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01–0.10% w/v.     

Live food production using batch culture and chemostat systems in Kuwait

A comparison of live food production using batch andchemostat systems between the facilities initiated in1988 and the re-establishment of new larger chemostatsystems, after Kuwaits liberation, is made. The livefood production dynamics achieved during the 1995–1996 spawning season of Shaem (Acanthopagrus latus)are discussed. Growth rate of the rotifers (Brachionusplicatilis) treated 24 h with a mixture of marinealgal species (Nannochloropsis, Isochrysis,Tetraselmis, Chlorella) plus other enrichment mediasuch as Super Selco, Protein Selco and Sanders priorto being supplied to the marine fish larvae isdiscussed. Rotifer production reached up to 2000 ×10⁶ individuals per day.     

Robust parameter estimation during logistic modeling of batch and fed‐batch culture kinetics

Methods for robust logistic modeling of batch and fed‐batch mammalian cell cultures are presented in this study. Linearized forms of the logistic growth, logistic decline, and generalized logistic equation were derived to obtain initial estimates of the parameters by linear least squares. These initial estimates facilitated subsequent determination of refined values by nonlinear optimization using three different algorithms. Data from BHK, CHO, and hybridoma cells in batch or fed‐batch cultures at volumes ranging from 100 mL–300 L were tested with the above approach and solution convergence was obtained for all three nonlinear optimization approaches for all data sets. This result, despite the sensitivity of logistic equations to parameter variation because of their exponential nature, demonstrated that robust estimation of logistic parameters was possible by this combination of linearization followed by nonlinear optimization. The approach is relatively simple and can be implemented in a spreadsheet to robustly model mammalian cell culture batch or fed‐batch data. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Comparative stability of ethanol production by Escherichia coliKO11 in batch and chemostat culture

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded. Received 13 January 1999/ Accepted in revised form 23 April 1999     

Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ana A. Arteni and Lu-Ning Liu equally contributed to the work.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Mixed culture kinetics of stringent and relaxed Escherichia coli cells in glucose-limited chemostat

The mixed culture kinetics of stringent and relaxed Escherichia coli cells were investigated in a glucose-limited chemostat at different dilution rates. Independent of the dilution rate the stringent cells competed out the relaxed cells. But the number of generations necessary for displaying the relaxed cells by the stringent ones increased with increasing dilution rate. The results are discussed as a consequence of the regulatory role of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) which is known to be present at different concentrations in stringent and relaxed cells under conditions of nutrient limitation. In addition, it is postulated that the coefficient of the maintenance metabolism according to PIRT (1965) is slower in stringent cells than in relaxed cells of E. coli.     

The role of hydrogen mass transfer for the growth kinetics of Methanobacterium thermoautotrophicum in batch and chemostat cultures

Hydrogen concentration was determined in batch and chemostat cultures of Methanobacterium thermoautotrophicum, both in the headspace and in the medium using mass spectrometry. The calculated dissolved hydrogen concentration in the medium as derived from the headspace hydrogen concentration when equilibrium conditions between gas and liquid phase were assumed, was ten times higher than the experimentally determined hydrogen concentration. Variation of the partial pressure of hydrogen resulted in different values for substrate affinity for hydrogen (K_s) and yield (Y) of the cells. Upon hydrogen limitation, K_s decreased while the yield coefficient for hydrogen increased, indicating a change in the affinity of the cells towards hydrogen. Received 15 November 1996/ Accepted in revised form 21 July 1997     

Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture

Sugar-limited batch growth of Dioscorea deltoidea and Catharanthus roseus plant cell cultures was studied in a 14-L stirred tank fermentor. With dissolved oxygen concentration monitored and maintained at nonlimiting levels, growth rates and ratios of dry weight to fresh weight were found to be strongly influenced by sugar concentration. Linear relationships between respiration rate and growth rate were observed, and respiration rate was found to drop to a maintenance level after sugar had been fully depleted from the medium. Diosgenin biosynthesis by D. deltoidea was shown to be independent of growth rate. Ajmalicine biosynthesis in C. roseus was negligible during sugar-limited growth but was induced by inoculation into an 80-g/L glucose solution.     

Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture

The influence of physiological parameters such as carbon substrate flux and O₂ uptake rates on energy metabolism are reported with reference to biomass productivity in whey chemostat culture. The combined results show that oxidoreductive energy metabolism may be attained independently of the yeast reaching its maximum respiratory capacity. A novel metabolic interpretation is presented proposing that a relative imbalance between glycolysis and subsequent oxidative steps alone is sufficient to account for the observed results. By means of a mathematical model the results could be reproduced under all experimental conditions. The new interpretation provides an insight into the manner in which energy mettbolism is regulated and influences growth-related process Kluyveromyces marxianus, as well as other yeasts with similar physiological characteristics. Correspondence to: J. I. Castrillo     

The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae

The amino acid sequences of cytochrome c₅₅₃ from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P₇₀₀ electron acceptor complex.