Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.     

Increased Growth of the Microalga Chlorella vulgaris when Coimmobilized and Cocultured in Alginate Beads with the Plant-Growth-Promoting Bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.     

Possibilities of bioconversion of agricultural waste with the use of microalgae

A new methodology of biological treatment and conversion of farm waste (manure and wash water) with the use of intensively cultivated phototrophic microorganisms (microalgae) is reviewed. Criteria for selection of microalgae and peculiarities of their intensive cultivation for efficient removal of biogenic elements from and destruction of the organic components of the wastes as well as the possibilities of cost-effective utilization of the resulting microalgal biomass are considered. Advantages and drawbacks of the new methodology are compared with those of conventional anaerobic techniques. Special attention is paid to the integrated technologies combining the aerobic conversion methods with microalgal post-treatment.     

Immobilization of cyanobacteria and microalgae on polyethylenimine-based sorbents

Immobilization of phototrophic microogranisms: microalgae (MA) and cyanobacteria (CB) on polyethylenimine (PEI)-based sorbents was studied. For this purpose, 3 insoluble porous polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine and immobilization of PEI on the surface of styrene–divinylbenzene copolymer. The sorbent on the basis of cross-linked PEI was also alkylated with hexadecyl bromide to achieve hydrophobicity of its surface. The analysis of kinetics and efficiency of immobilization assessed for the model MA and CB cultures revealed the significant difference in the sorption activity of different types of sorbents depending on their synthesis procedure, chemical composition and hydrophilic-hydrophobic properties of polymeric surface. The hydrophobic sorbent obtained by immobilization of PEI on the surface of styrene–divinylbenzene copolymer characterized by very low sorption activity towards CB and MA cells. The highest immobilization efficiency of phototrophic cells was achieved for the hydrophilic sorbent on the basis of PEI cross-linked with epichlorohydrine, which provided the attachment of 50–70% of cells during 3 h of incubation. The hydrophobic sorbent based on alkylated cross-linked PEI effectively immobilized CB cells, while the colonization of the polymer surface by MA cells was very scarce. The noticed effect is explained by difference in prokaryotic (CB) and eukaryotic (MA) types of surface structures organization. Assessment of photosynthetic activity of immobilized MA cells by pulse-modulated fluorometry showed that hydrophobic sorbents had no toxic effect on the cells, while toxicity of hydrophilic cross-linked PEI-based sorbent was observed only after long-term cultivation ofphototrophic cells with this sorbent.     

Rhodococcus sp. F92 immobilized on polyurethane foam shows ability to degrade various petroleum products

This work reports on the immobilization and performance of a hydrocarbon-degrading microorganism on polyurethane foam (PUF) in the bioremediation of petroleum hydrocarbons. The ability of four different microorganisms to immobilize on PUF and to degrade various petroleum products (Arabian light crude (ALC), Al-Shaheen crude (ASC), diesel and oil slops) was assessed by measuring the n-alkane fraction remaining in the petroleum products over time. A Rhodococcus sp. (designated as F92) had the highest number of immobilized viable cells (10(9) cells per cm3 PUF) and a maximum attachment efficiency of 90% on PUF of a density of 14 kg/m3. Scanning electron microscopy showed the presence of extracellular structures that could play an important role in the immobilization of F92 on PUF. Analysis by GC-MS revealed that both free and immobilized F92 cells were able to degrade approximately 90% of the total n-alkanes in the petroleum products tested within 1 week at 30 degrees C. Rhodococcus sp. F92 was efficiently immobilized onto PUF and the immobilized cells were able to degrade a variety of petroleum products such as ALC, ASC, diesel and oil slops. The results suggest the potential of using PUF-immobilized Rhodococcus sp. F92 to bioremediate petroleum hydrocarbons in an open marine environment.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.     

Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review

Microalgae have the ability to mitigate CO₂ emission and produce oil with a high productivity, thereby having the potential for applications in producing the third-generation of biofuels. The key technologies for producing microalgal biofuels include identification of preferable culture conditions for high oil productivity, development of effective and economical microalgae cultivation systems, as well as separation and harvesting of microalgal biomass and oil. This review presents recent advances in microalgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production. The effects of different microalgal metabolisms (i.e., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth), cultivation systems (emphasizing the effect of light sources), and biomass harvesting methods (chemical/physical methods) on microalgal biomass and oil production are compared and critically discussed. This review aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.     

MICROALGAL BIOTRANSFORMATION OF STEROIDS1

Microbial biotransformation of steroids is not a new concept, but most studies in this field have focused on fungal and bacterial systems. Microalgae, despite their photosynthetic ability and immense biodiversity, have not received much attention in this aspect until recently. Since the publication of the first article on microalgal biotransformation of steroids about 20 years ago, there have been many reports describing different modifications, including hydroxylation, reduction, side‐chain degradation, and isomerization introduced by these microorganisms on estrane, androstane, and pregnane derivatives. On the other hand, the development of new large‐scale cultivation systems, the adaptation of existing fermentation techniques to microalgae, and the introduction of microalgal genetic manipulation methods have made these organisms promising candidates for a wide range of biotechnological processes, including biotransformations. In this review, we have summarized the steroid transformation patterns of several microalgal strains and present a perspective of the future trends in microalgal biotechnology, including the possibility of adapting relatively new techniques, such as organic media catalysis and cell immobilization, to this specific field.     

Biofiltration--the treatment of fluids by microorganisms immobilized into the filter bedding material: a review

Biofiltration is distinguished from other biological waste treatments by the fact that there is a separation between the microorganisms and the treated waste. In biofiltration systems the microorganisms are immobilized to the bedding material, while the treated fluid flows through it. In recent decades, a vast amount of literature has been written on single experiments involving the treatment of fluids by immobilized microorganisms. Several artificial immobilization methods have been examined and impressive results have been achieved in the treatment of fluids with one of the artificial immobilization methods the entrapment of microorganisms within polymer beads. This method, even though it needs to be improved, seems to have a future potential in commercial biofiltration systems. The methods of artificial immobilization of microorganisms within biofiltration systems have several advantages, but also suffer from several disadvantages in comparison to the treatment of fluids by naturally attached microorganisms. Understanding the mechanisms and forces responsible for the attachment of microbes to the bedding material, in attempt to improve this attachment, is of the utmost importance. Further improvement of the artificial entrapment of microorganisms within polymers will allow the exploitation of the advantages of this method in the treatment of fluids. The aim of this review essay is to introduce the main principles of two immobilization processes - the self-attachment of microorganisms to the bedding material and the artificial entrapment of microorganisms within polymer beads. Both treatments of liquids and gases with each immobilization process are discussed. The advantages and disadvantages of each immobilization process are pointed out and different aspects of the fluid treatment with the two immobilization processes are compared.     

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.     

The enzymatic conversion of L-histidine to urocanic acid by whole cells of Micrococcus luteus immobilized on carbodiimide activated carboxymethylcellulose.

Whole cells of Micrococcus luteus (formerly Sarcina lutea ATCC 9341) have been covalently linked to a carboxymethylcellulose support system, with the retention of histidine ammonia-lyase activity. The dependence of the rate of urocanic acid formation on pH, temperature, and added surfactant concentration was similar for the free and the immobilized cells. The immobilization procedure used is based on the carbodiimide activation of carboxymethylcellulose and has been optimized for the histidine ammonia-lyase activity of the immobilized cells on a given weight of cellulose. In a column reactor at 23 degrees C and superficial velocity of 0.044 cm/min, 5 g of cellulose with bound cells gave a 35% conversion of an L-histidine solution (0.25M, pH 9.0) to urocanic acid for 16 days of continuous operation. The scope of this carbodiimide assisted immobilization procedure has been investigated for a series of microorganisms and a variety of carboxylate functionalized supports.     

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Plasmid maintenance and physiology of a genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> strain during continuous <Emphasis Type="SmallCaps">l</Emphasis>-carnitine production

The effect of immobilization on cell physiology and how this determines cell metabolic performance is an important concern for developing bioprocess. This is particularly true for genetically modified microorganisms and their genetic stability. For this reason the stability and physiological state of plasmid-bearing E. coli cells were ascertained by flow cytometry. Differences in the cellular DNA and protein content (15-20%) permit discrimination of control and plasmid-bearing cells, as well as adaptation to continuous cultivation conditions in both freely suspended and immobilized states to be monitored. Moreover, the observed metabolic burden due to maintenance and over-expression of plasmid-coded genetic material and slow cell growth in poorly-viable immobilized cells were found to be the main factors contributing to strain stabilization.     

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi was developed. The suitable conditions for pellet formation by filamentous fungi were determined. Among the strains tested, Trichoderma reesei QM 9414 showed superior pellet forming ability. Its pellets were used to harvest oleaginous microalga Scenedesmus sp. With increasing volume ratio of fungal pellets to microalgae culture up to 1:2, >94% of microalgal cells were rapidly harvested within 10 min. The ratio of fungal pellets could manipulate both harvesting time and initial concentration of microalgal cells in the pellets. The microalgae–fungal pellets were successfully used as immobilized cells for effective phytoremediation of secondary effluent from seafood processing plants under nonsterile condition. The chemical oxygen demand, total nitrogen, and total phosphorus removal were >74%, >44%, and >93%, respectively. The scanning electron microscopy showed that the microalgal cells were not only entrapped in the pellets but also got attached to the fungal hyphae with sticky exopolysaccharides, possibly secreted by the fungi. The extracted lipids from the pellets were mainly composed of C16–C18 (>83%) with their suitability as biodiesel feedstocks. This study has shown the promising strategy to rapidly harvest and immobilize microalgal cells and the possible application in phytoremediation of industrial effluent.     

Immobilization of microorganisms with PVA hardened by iterative freezing and thawing

In order to utilize polyvinyl alcohol (PVA) as a gel matrix for the immobilization of microorganisms, PVA was subjected to iterative freezing-thawing. The effects of the procedure on the mechanical characteristics of the PVA hydrogel and the stability of the immobilized microorganisms were investigated. PVA showed rubber-like elasticity after iterative freezing-thawing. Gel strength increased with the iteration number of freezing-thawing until seven iterations. Although the activities of both the free and immobilized cells decreased during the iteration of freezing-thawing, addition of cryoprotectants such as glycerol and skim milk was effective for preventing the decrease in activity.     

Biodegradation of hydrocarbon contamination by immobilized bacterial cells

This study examined the capacity of immobilized bacteria to degrade petroleum hydrocarbons. A mixture of hydrocarbon-degrading bacterial strains was immobilized in alginate and incubated in crude oil-contaminated artificial seawater (ASW). Analysis of hydrocarbon residues following a 30-day incubation period demonstrated that the biodegradation capacity of the microorganisms was not compromised by the immobilization. Removal of n-alkanes was similar in immobilized cells and control cells. To test reusability, the immobilized bacteria were incubated for sequential increments of 30 days. No decline in biodegradation capacity of the immobilized consortium of bacterial cells was noted over its repeated use. We conclude that immobilized hydrocarbon-degrading bacteria represent a promising application in the bioremediation of hydrocarbon-contaminated areas.     

Chlorella sorokiniana immobilized on the biomatrix of vegetable sponge of Luffa cylindrica: a new system to remove cadmium from contaminated aqueous medium

A new sorption system of microalgal cells immobilized on the biostructural matrix of Luffa cylindrica for sequestering cadmium is reported. Free and immobilized Chlorella sorokiniana removed cadmium from 10 mgl(-1) solution at the efficiency of 92.7% and 97.9% respectively. Maximum cadmium sorption was observed to be 39.2 mgg(-1) at equilibrium (C(eq)) of 112.8 mgl(-1) by immobilized microalgal biomass as compared to 33.5 mgg(-1) at C(eq) of 116.5 mgl(-1) by free biomass from initial concentration of 150 mgl(-1). In continuous liquid flow column, the cadmium sorption capacity of immobilized C. sorokiniana was 192 mgg(-1), which was 73.2% of the total metal passed in 51.5 l. Metal desorption with 0.1 M HCl was 100% and the desorbed immobilized system was reusable with a similar efficiency in the subsequent cycle.     

Immobilization of Saccharomyces yeast cells on electret polyethylene films

The method of electret-thermal analysis developed in dielectric physics has been applied to monitor bioelectric phenomena that accompany the immobilization of microorganisms on electret substrates. A spectrum of thermo stimulated currents for biofilms consisting of immobilized Saccharomyces cells has been obtained. The spectrum represents a halo in the region of 30-90 degrees C. The immobilization of Saccharomyces cells on electret polyethylene films results in their depolarization that is recorded on the spectra of thermostimulated currents as a reduction of the current peak corresponding to the polyethylene melting point. A hypothesis has been put forward that explains the phenomenon by the absorption of the electric energy of polarized substrates by the cells for the occurrence of metabolic reactions.     

Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology

Abstract

This work focuses on the immobilization of a crude inulinase extract obtained by solid-state fermentation using spray-drying technology. Maltodextrin and arabic gum were used as immobilizing agents. The effects of inlet air temperature, maltodextrin/arabic gum ratio and mass fraction of crude enzyme extract on the activity of immobilized inulinase were assessed using a central composite rotatable design (CCRD) (2³). The optimum operational conditions for the immobilization of inulinase by spray-drying was obtained at an inlet air temperature of 200°C, mass fraction of crude enzyme extract of 0.5 wt% and using only arabic gum as immobilizing agent. The immobilized enzyme had good thermostability, comparable with other inulinases obtained from different microorganisms. The method used gave good enzyme activity after immobilization and could be applied to other enzymes which have good thermal stability.