Immobilization of Botryococcus braunii in alginate: influence on chlorophyll content,photosynthetic activity and degeneration during batch cultures

Summary Immobilization of the hydrocarbonrich microalga Botryococcus braunii in calcium alginate beads results in a large increase in chlorophyll content and chlorophyll photosynthetic activity, relative to free cells, at any stage of standard batch cultures. Immobilization exerts a protective influence on ageing cultures both under standard and air lift conditions. Decreases in chlorophyll content and photosynthetic activity are delayed and slowed down; the organization of protein-chlorophyll complexes is stabilized. These positive effects are related to protection of entrapped cells against photoinhibition owing to gel screening and self-shadowing.Entrapped cells show also, immediately after immobilization, a higher photosynthetic activity than free controls. The unusually high activity yield thus achieved probably results from increase in ionic concentration in the microenvironment of B. braunii.     

Growth and hydrocarbon production ofBotryococcus braunii immobilized in calcium alginate gel

Summary The colonial microalgaB. braunii, immobilized in calcium alginate beads, shows active photoautotrophic growth. Nevertheless, the rates of increase in cell number and, to a lesser extent, in biomass are substantially lower when compared to free cultures. Such features are related to steric contraints which occasion also the formation of large spherical colonies in the gel, showing an unsual mulberry organization. Some cracks due to the development of underlying colonies appear at the surface of the beads. Alga release remains low, however, during the cultures. EntrappedB. braunii retain the ability to produce extracellular hydrocarbons; the structure of the latter is not affected by immobilization but their relative abundances can undergo some variations. Entrapment leads to marked improvements in hydrocarbon production; decrease in growth rates is therefore associated, in alginate gel, with a still more pronounced diversion ofB. braunii metabolic activity towards hydrocarbon generation. It appears also that the improvements in hydrocarbon production, due to strain selection and to culture condition adjustment, obtained in free cultures, can be directly applied toB. braunii immobilized in alginate beads.     

Removal of nitrate from water by foam-immobilizedPhormidium laminosum in batch and continuous-flow bioreactors

Cells of the non-N₂-fixing cyanobacteriumPhormidium laminosum were immobilized in polyurethane (PU) foams either by absorption or by entrapment in the PU prepolymer followed by polymerisation and by adsorption onto polyvinyl (PV) foams. Although entrapment caused toxicity problems which lead to rapid death of the immobilized cells, they were immobilized successfully by adsorption onto PU or PV foams and maintained their photosynthetic electron transport activities (PS I, II, I + II) for at least 7 weeks. Changes in the morphology resulting from immobilization, as revealed by scanning electron microscopy (SEM) and low temperature-SEM, were investigated. Batch cultures and a continuous-flow packed bed photobioreactor were used to study nitrate removal from water. The effects of light intensity and CO₂ concentration on bioreactor performance were studied with respect to the nitrate uptake efficiency of the system. It was concluded thatP. laminosum immobilized on polymer foams is of potential value for biological nitrate removal in a continuous-flow system. author for correspondence     

BACTERIAL INFLUENCE UPON GROWTH AND HYDROCARBON PRODUCTION OF THE GREEN ALGA BOTRYOCOCCUS BRAUNII1

Batch cultures of the hydrocarbon-rich alga Botryococcus braunii, Kütz. (axenic strains, non-axenic strains, associations with selected microorganisms) were examined with regard to total biomass and hydrocarbons at the onset of the stationary phase. Pronounced variations, related to the origin of the strains and to growth conditions, were observed with axenic cultures. It also appeared that the presence of microorganisms is not essential for high hydrocarbon production. Nevertheless, numerous bacteria were shown to exert considerable influence, antagonistic or beneficial, on B. braunii growth yield and hydrocarbon production. Such effects were strongly dependent on the species involved and on culture conditions. The presence of various microorganisms can influence not only the quantity of hydrocarbons produced, but also their level in the algal biomass and their relative abundance. However, their chemical structure is not affected. Intricate relationships were observed in B. braunii-bacteria systems and numerous factors (including, in some cultures, large positive effects due to bacterially produced CO₂) were implicated. Accordingly, specific associations should provide appropriate conditions for renewable hydrocarbon production via B. braunii large scale cultures.     

Modelling the microenvironment of a lipase immobilized in polyurethane foams

The effects of partition of substrates and product on the modelling of the microenvironment of an immobilized lipase were evaluated using Response Surface Methodology. The esterification of butyric acid with ethanol in n-hexane, catalyzed by Candida rugosa lipase immobilized in two biocompatible and relatively hydrophilic polyurethane foams (“Hypol FHP 2002™” and “Hypol FHP 5000™”) was used as the model system. For each set of initial conditions, the final concentration of substrates and ethyl butyrate in the microenvironment, at equilibrium, Cmicro, were estimated by mass balancing bulk and foams. The Cmicro values obtained were used to estimate the corresponding partition coefficients of ethanol (P_EtOH), butyric acid (P_BA) and ester (P_EB), between the foams (microenvironment) and the bulk medium. Foams containing previously inactivated lipase, as well as lipase-free foams were used. For both substrates, Cmicro values were, in the majority of the experiments, higher than their macroenvironmental counterparts. The lowest Cmicro values were observed with the less hydrophilic foam (“FHP 5000”). A decrease of Cmicro_EtOH in both foams and Cmicro_BA in “FHP 5000” foams, was obtained upon lipase immobilization. P_EB values were, in all cases, close to zero. This is beneficial in terms of the shift in reaction equilibrium, product recovery and alleviation of product inhibition effects.     

Activity and batch operational stability of Candida rugosa lipase immobilized in different hydrophilic polyurethane foams during hydrolysis in a biphasic medium

The aim of this study was to investigate eventual relationships between some physico-chemical properties (e.g. porosity, aquaphilicity, partition coefficient for oleic acid and drying curves) of relatively hydrophilic polyurethane foams and the activity and batch operational stabiliy of Candida rugosa lipase immobilized within these foams. Two biocompatible polyurethane pre-polymers ("HYPOL FHP 2002^TM" and "Hypol FHP X4300^TM" from Hampshire Chemical GmbH, Germany) were tested as immobilization supports. The model reaction was the hydrolysis of crude olive residue oil in a biphasic aqueous/n-hexane medium. Drying curves under normal and reduced pressures suggested that water molecules are more strongly bound to the "FHP 2002" than to "FHP X4300" foams. This is in agreement with the higher aquaphilicity value estimated for the "FHP 2002" foam (3.7 vs 2.8). For every enzyme loading tested, hydrolysis efficiency was considerably higher for the lipase in "FHP X4300" foam when compared to the other counterpart. However, internal mass transfer limitations seem to be more severe with "FHP X4300" foams. Operational stability was evaluated in 10 consecutive batches (1 batch = 23 hours) for both immobilized preparations. A fast deactivation was observed for both biocatalysts. However, a slightly higher operational stability was observed for the lipase in "FHP 2002" foam. For the lipase in "FHP X4300" foam, the activity decay can be explained by a dramatic lipase leakage from the foam observed along successive batches. For the lipase in "FHP 2002" foam, no significant enzyme loss was observed along the reutilizations probably due to a higher number of multi-point attachment between the lipase and its support. In fact, activity and operational stability of Candida rugosa lipase in "FHP 2002" and "FHP X4300" foams appear to be related with the strength and/or the number of covalent binding between the enzyme and the support rather than to the physico-chemical properties evaluated in this work.     

Lipid and hydrocarbon production byBotryococcus spp. under nitrogen limitation and anaerobiosis

The production of lipids and hydrocarbons in batch cultures of the algaeBotryococcus braunii andB. protuberans has been studied with respect to nitrogen limitation under aerobic and anaerobic conditions. Nitrogen deficiency significantly decreased the dry weight, chlorophylla and protein contents but the amounts of carotenoids, carbohydrates and lipids increased in both the species. Nitrogen starvation gave a 1.6-fold increase in lipid content. Anaerobiosis under nitrogen deficient conditions gave greater lipid production than anaerobiosis in nitrogen supplemented medium. Under nitrogen deficiency, the hydrocarbon fraction increased and the polar lipids decreased. Anaerobiosis induced hydrocarbon synthesis more significantly than nitrogen deficiency but decreased other non-polar and polar lipids.     

Culture of the green microalga Botryococcus braunii Showa with LED irradiation eliminating violet light enhances hydrocarbon production and recovery

The green microalga Botryococcus braunii (B. braunii), race B, was cultured under light-emitting diode (LED) irradiation with and without violet light. This study examined the effect of violet light on hydrocarbon recovery and production in B. braunii. C34 botryococcene hydrocarbons were efficiently extracted by thermal pretreatments at lower temperatures when the alga was cultured without violet light. The hydrocarbon content was also higher (approximately 3%) in samples cultured without violet light. To elucidate the mechanism of effective hydrocarbon recovery and production, we examined structural components of the extracellular matrix (ECM). The amounts of extracellular carotenoids and water-soluble polymers extracted by thermal pretreatment from the ECM were decreased when the alga was cultured without violet light. These results indicate that LED irradiation without violet light is more effective for hydrocarbon recovery and production in B. braunii. Furthermore, structural ECM components are closely involved in hydrocarbon recovery and production in B. braunii.     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Some effects of nitrogen-starvation on nitrogen and carbohydrate metabolism inAnkistrodesmus braunii

Enzymic activities have been measured in cell-free extracts from nitrogen-starved cultures ofAnkistrodesmus braunii. During ten hours of nitrogenstarvation the activities of the enzymes nitrite reductase (E.C.1.6.6.4), glutamic dehydrogenase (E.C.1.4.1.4), glutamine synthetase (E.C.6.3.1.2) and urea amidolyase (E.C.3.5.1.5) were derepressed while the activities of the enzymes malate dehydrogenase (E.C.1.1.1.37) and hexokinase (E.C.2.7.1.1) remained more or less unchanged. In contrast, the photosynthetic capacity of the nitrogen-starved cultures declined rapidly and accompanying this decline were losses in the activities of ribulose diphosphate carboxylase (E.C.4.1.1.39) and triose phosphate-NADP-dehydrogenase (E.C.1.2.1.13).     

A comparison of two techniques (adsorption and entrapment) for the immobilization of Aspergillus niger in polyurethane foam

Summary Aspergillus niger was immobilized by adsorption and entrapment in polyurethane foams and the efficiency of retention capacity, citric acid productivity and the operational stability of a fluidized bed reactor were then compared. The adsorption technique was superior to the entrapment technique, and it was possible to obtain bioparticles capable of keeping their activity for more than 25 days.     

Genome size and phylogenetic analysis of the A and L races of <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. There are three different races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Each race also has many strains that are distinguished by the location from which they were collected. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. To begin to address this problem, we determined the genome size of the A race, Yamanaka strain, and the L race, Songkla Nakarin strain, of B. braunii. Flow cytometry analysis indicates that the A race of B. braunii has a genome size of 166.0 ± 0.4 Mb, while the L race has a substantially larger genome size at 211.3 ± 1.7 Mb. We also used phylogenetic analysis with the nuclear small subunit (18S) rRNA gene to classify strains of the A and B races that have not yet been compared evolutionarily to previously published B. braunii phylogenetics. The analysis suggests that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce.     

Mechanism of non-isoprenoid hydrocarbon biosynthesis in Botryococcus braunii

The green unicellular alga Botryococcus braunii shows unusually high concentrations of non-isoprenoid very long chain hydrocarbons. The structure of such hydrocarbons, the relative efficiency of various long chain fatty acids as precursors, the relationship between fatty acid and hydrocarbon concentrations (over the different physiological stages of the alga achieved during batch cultures) and the preferential localization of fatty acids lead to the conclusion that all the major non-isoprenoid hydrocarbons of B. braunii derive from the same direct precursor, oleic acid. Feeding experiments, using doubly labelled oleic acid, show that the whole carbon chain of the latter is incorporated into final hydrocarbons; accordingly such compounds do not originate from a head-to-head condensation mechanism with oleic acid acting as donor. Various features (regarding chiefly the systematic occurrence of a terminal double bond in B. braunii hydrocarbon, their close specific activities after feeding and the large inhibition in their production achieved using dithioerythritol) show that the biosynthesis of B. braunii hydrocarbons probably takes place via an elongation-decarboxylation mechanism related to that operating in some higher plants.     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

CHEMICAL PROFILE OF SELECTED SPECIES OF MICROALGAE WITH EMPHASIS ON LIPIDS1

The lipid profile of seven species of unicellular eukaryotic microalgae grown under controlled conditions was studied with emphasis on the hydrocarbons and the fatty acids as part of a search for oil-producing algae. Green, slow-growing colonies of Botryococcus braunii Kutz contained the highest lipid content of 45% based on the organic weight, with an increase to 55% under nitrogen deficiency and with no effect of sodium chloride stress. Ankistrodesmus sp. Thomas, Dunaliella spp., Isochrysis sp., Nannochloris sp. Thomas, and Nitzschia sp. Chapman contained an average of 25% lipids under nitrogen sufficient conditions. Nitrogen deficiency resulted in significant increase in the lipid content in all species but Dunaliella spp., which produced a higher content of carbohydrates. Significant low amounts of acyclic hydrocarbons were detected only in Botryococcus braunii Kutz and not in the other algae. The major hydrocarbon fractions in nitrogen deficient Botryococcus braunii Kutz, Dunaliella salina Thomas, Isochrysis spp. and Nannochloris sp. Thomas were cyclic and branched polyunsaturated components which were identified as various isoprenoid derivatives. The polar lipid composition of glycolipids and phospholipids of all species investigated was fairly typical of photosynthetic eukaryotic algae. Fatty acid composition was species specific, with changes occurring in the relative amounts of individual acids of cells cultivated under different conditions and growth phases. All species synthesized C14:0, C16.0, C18:1, C18:2 and C18:3 fatty acids; C 16:4 in Ankistrodesmus sp. Thomas; C18:4 and C 22.6 in Isochrysis sp.; C16:2, C16:3 and C20:5 in Nannochloris sp. Thomas; C16:2, C16:3 and C20:5 in Nitzschia sp. Chapman. Nitrogen deficiency and salt stress induced accumulation of C18:1 in all treated species and to a lesser extent in Botryococcus braunii Kutz. The low production of hydrocarbons under optimal growth conditions and the high production of hydrocarbons under limited growth conditions cannot support the notion that microalgae can be utilized as biosolar energy converters for the production of liquid fuel, but point to the availability of a variety of neutral and polar lipid products.     

Photosynthesis and glycoliate excretion by immobilizedChlorella emersonii

Summary Undiminished capacities for photosynthetic oxygen evolution and glycollate excretion were obtained over a 6 month period after the immobilization ofChlorella emersonii by entrapment in calcium alginate gel. The activities of enzymes of photosynthesis and glycollate metabolism also remained unchanged over this period.     

Epoxidation of propylene utilizing Nocardia corallina immobilized by gel entrapment or adsorption

Nocardia corallina B-276 cells are capable of catalysing the direct epoxidation of propylene to propylene oxide, through a monooxygenase enzyme system. The present work was undertaken to see how immobilization of the whole cells by adsorption or entrapment on solid supports would influence the rate and duration of the epoxidation activity. With immobilization by adsorption, the propylene oxide forming activity was highest on hydrophobic supports, such as polypropylene or polyethylene. Under certain conditions the activity was three times that of the free control cells. However, the duration of epoxidation activity was considerably less for the adsorbed cells. The cell loading by adsorption, determined with¹⁴C-labelled cells was 1.0–2.8 mg dry cell weight g^?1of support. The cells, whether immobilized or not (controls), were tested using a continuous gas flow packed-bed or bubble-type reactor. Immobilization of the cells by entrapment in calcium alginate beads gave about the same propylene oxide forming activity and stability as with control cells, provided the reactor was operated at low temperature (30°C) and low oxygen content (20%) of the feed stream. The results suggest that entrapment in a hydrophobic matrix might be a more favourable system; although additional investigation of the rate limiting steps as well as the cause of the activity loss with time is needed.     

The biosynthesis of long-chain hydrocarbons in the green alga Botryococcus braunii

The green colonial alga Botryococcus braunii has unusually high levels of hydrocarbons. Two distinct sites of hydrocarbon accumulation are present in the species: an internal pool present in cytoplasmic inclusions and an external pool in the trilaminar outer walls and associated globules. It is generally assumed that the hydrocarbons are produced within the cells and then excreted into the external pool to maintain the intracellular content at a normal value. Various feeding experiments showed, however, that the radioactivity of the external pool is much higher than the internal one. On the other hand, there was no decrease in the labelling of internal hydrocarbons in chase experiments. Therefore, an excretory process apparently does not take place in B. braunii. The outer wall, therefore, is the main site of hydrocarbon accumulation and also the place where the bulk of B. braunii hydrocarbons are produced. The outer wall also is involved in the matrix of colony formation and the above findings account for the sharp decrease of hydrocarbon production which is associated with the loss of colonial habit. The cultures were also shown to be unable, under usual growth conditions, to catabolize their own hydrocarbons. Such a feature, along with the extracellular location of the main site of production, may account for the abnormally high content of hydrocarbons typical of B. braunii.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.