Loofa (Luffa cylindrica) sponge: Review of development of the biomatrix as a tool for biotechnological applications

The review discusses the development of loofa sponge (Luffa cylindrica) as a biotechnological tool and the diversity of applications in which it has been successfully used since it was first reported as a matrix for the immobilization of microbiological cells in 1993. The fibro‐vascular reticulated structure, made up of an open network of random lattices of small cross‐sections coupled with very high porosity (79–93%), having very low density (0.02–0.04 g/cm³), and high specific pore volume (21–29 cm³/g), has the characteristics of a carrier/scaffold well‐suited for cell immobilization. This has been confirmed through the immobilization of cells of diverse types, including filamentous and microalgae, fungi, bacteria, yeasts, higher plants, and human and rat hepatocytes. The cells immobilized in loofa sponge have performed well and better than free suspended cells and those immobilized in conventionally used natural and synthetic polymeric materials for the production of ethanol, organic acids, enzymes, and secondary metabolites. The loofa‐immobilized cell systems have been efficiently used for the treatment of wastewaters containing toxic metals, dyes, and chlorinated compounds, and the technology has been used to develop biofilms for the remediation of domestic and industrial wastewaters rich in inorganic and organic matter. In addition, three‐dimensional loofa sponge scaffolds for hepatocyte culture have been suggested to have the potential for development into a bioartificial liver device. Loofa sponge is a cost‐effective, eco‐friendly, and easy to handle matrix that has been used successfully as a biotechnological tool in a variety of systems, purposes, and applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:573–600, 2013     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

THE USE OF IMMOBILIZATION IN ALGINATE BEADS FOR LONG-TERM STORAGE OF PSEUDANABAENA GALEATA (CYANOBACTERIA) IN THE LABORATORY1

A new method for long-term storage of algal cultures in the laboratory was tested. The procedure is based on the cell immobilization technique. Cells of the filamentous cyanobacterium Pseudanabaena galeata Bocher were immobilized in sodium-alginate beads and stored for 14–18 months. The structure and functional features of the organism were maintained in this immobilized state and no ultrastructural biochemical, or growth rate differences were detected between stock and previously immobilized cell cultures after this period. We discuss the advantages of this method compared to other preservation methods and recommend the use of immobilization in alginate beads for long-term algal storage.     

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.     

Studies on Bacterial Cell Multiplication

Chemical composition of the filamentous cells of Lactobacillus delbrueckii produced by vitamin B₁₂ deficient culture was studied. Protein and RNA contents per unit cell volume of the filamentous cells were nearly equal to those of normal cells, but the DNA content was much reduced. A cytoplast of the filamentous cell possessed about twice as large volume as that of the normal cell. A cytoplast of either filamentous or normal cell seems to contain the same amount of DNA. DNA level and membrane formation necessary for cell division remained as future problems.     

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.     

Expression of Surface Coat Glycoconjugates by Bacteria-Fed Tetrahymena1

ABSTRACT. We have shown that bacteria-fed Tetrahymena express at their surface and excrete into the medium a glycoconjugate absent in axenically grown cells. A preliminary analysis of the purified molecule is given. Immunolabeling of blotted surface extracts and fixed cells shows that bacteria-fed Tetrahymena build up a surface coat whose material originates totally or in part in the mucocysts. The glycoconjugate is located externally on the coat and mediates cell immobilization and immunolabeling by the serum. The results also indicate that axenic cells are probably devoid of surface coat.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Immobilization of yeast cells in plant cell wall frameworks

Summary Cell-structured support materials (CSM) representing the cell framework of denaturated and extracted mosses, duckweeds or parenchyma tissue particles have been used for the immobilization of Saccharomyces cerevisiae cells. The method consists of inoculation by soaking the dehydrated materials in a yeast suspension and propagation of the yeast cells that reach the relatively closed inner volumes of the cell-structured particles (inter- or intracellular spaces). In spite of high cell densities (up to 2.5 × 10⁹ cells/g wet immobilizate) the velocity of microaerobic glucose consumption was little influenced by intraparticular diffusion resistances, when yeast loaded CSM made from Wolffia arrhiza was incubated in 100 mM glucose at room temperature.     

Activity of Some Aminopeptidases in Immobilized Cells of Daucus carota

Carrot cell suspension culture prior to immobilization by glutaraldehyde was permeabilised by Tween 80. The values of pH optimum for L-alanine aminopeptidase, L-proline iminopeptidase, and L-arginine aminopeptidase were 8.2, 7.4, and 7.9, respectively. The immobilized cells showed significantly lower aminopeptidase activity when compared to untreated cells. Alginate hydrogel was successfully used for immobilization of carrot cells retaining the activity of some aminopeptidases.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

Surface modified zeolite, a novel carrier material for Azotobacter chroococcum

A new immobilization matrix based on zeolite has been developed to immobilize Azotobacter chroococcum, for fixing nitrogen, with an intention to hold the cells in the root zone of the plants and to protect them under stressful conditions. The matrix has been developed by modifying the surface of the zeolite with surfactant. This enhances the hydrophobicity of the material and also modifies the surface charge, which in turn enhances the immobilization. Surface modified zeolite-A (SMZ-A) has been compared with commercial zeolite-A (CZA) for immobilization efficiency. CZA is non-toxic for A. chroococcum but is inefficient to adsorb the cells whereas SMZ-A showed 100% adsorption of the microbial cells wherein it was observed that for 1 l of broth culture with total viable count of 10⁸ cfu ml⁻¹ cells of A. chroococcum, a minimum dose of 0.7 g SMZ-A and minimum contact time of 10 h is required to achieve 100% adsorption. Adsorption was confirmed by the cell count and light as well as scanning electron microscopy. Most importantly, the cells adsorbed on SMZ-A could fix the atmospheric nitrogen up to 13 mg g⁻¹ sucrose consumed, which was comparable with the control (unadsorbed cells), which confirms the survival and nitrogen fixation activity of the bacteria. Responsible Editor: Euan K. James.     

Overexpression of ftsA induces large bulges at the septal regions inEscherichia coli

High-level expression offtsA, an essential cell division gene inEscherichia coli, inhibits cell septation and causes the formation of filaments that develop spherical bulges up to 4 m in diameter. These bulges may emanate from septation sites, since they were evenly spaced in relation to one another and to the cell poles. Electron microscopic examination of thin sections through the bulged regions reveals large electron-lucent, plate-like regions that appear to originate from the membrane. In addition, these bulging filamentous cells contain more hexosamine per mass than control cells. A model is proposed that the electron-lucent regions observed by electron microscopy are caused by peptidoglycan accumulation stimulated by the overexpression offtsA.     

SulA-independent filamentation of<Emphasis Type="Italic"> Escherichia coli</Emphasis> during growth after release from high hydrostatic pressure treatment

To improve the efficiency of sterilization by high hydrostatic pressure treatment (HPT), it is desirable to know the biochemical process of bacteria most sensitive to the treatment. We investigated growth properties after release from HPT of exponentially growing Escherichia coli K-12 cells. We observed growth retardation after treatment (30 min at 37°C) above 75 MPa. Long filamentous cells of about eight times normal cell length were observed at 90 min growth after treatment at 75 MPa. In the subsequent period the filamentous cells divided into normal-sized cells. recA and sulA mutant strains also formed filamentous cells, indicating that filamentation was SulA-independent. Nucleoids segregated normally in the filamentous cells. Only one FtsZ ring (or none) was detected at possible division sites in the elongated cells. Western blotting analysis demonstrated that the amount of FtsZ protein was not affected by the treatment. GTP-dependent in vitro polymerization of either FtsZ protein in E. coli crude extract or purified FtsZ protein, however, was sensitive to HPT. These facts suggest that HPT at 75 MPa denatures a fraction of FtsZ molecules, and that these denatured molecules interfere with the polymerization of functional FtsZ, resulting in the significantly reduced number of FtsZ rings.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

High density cultivation of Dictyostelium discoideum in a rotating polyurethane foam-bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In order to achieve high cell density cultivation, polyurethane foam (PUF) with high porosity was introduced as new matrix for the immobilization of D. discoideum. The results showed that about 88–93% cells of D. discoideum were adsorbed onto the PUF particles after 100 min equilibrium between adsorbed and free cells, and the highest immobilization rate was achieved by adding the same quantity of PUF matrix with the thin cylinder style. Furthermore, polyurethane foam was used as the immobilization matrix in a rotating PUF-bed bioreactor system. With batch cultures in the rotating bed bioreactor, the concentration of immobilized cells in the PUF carrier increased to 4.2 × 10⁷ cells ml⁻¹ after 167 h cultivation, which was about fourfold higher than the maximal cell density in the conventional free-cell culture. Further studies showed that the cells of D. discoideum were not just simply adsorbed on the surfaces, but actively attached to the surfaces through their network of pseudopodia or filopodia. The present work is very promising to improve the productivity of recombinant proteins in D. discoideum with high cell density in this novel rotating bed bioreactor.     

Modified hydroxyalkyl methacrylate gel as a support for immobilization of yeast cells

Summary Whole cells of Zygosaccharomyces lactis have been covalently linked to fine-grained hydroxyalkyl methacrylate gel Spheron P 1000 E which was prepared by treatment with epichlorhydrin and modified by an amine spacer. Experiments on the coupling of permeabilized and non-permeabilized cells to this gel support have shown that immobilized cell agregates may be obtained by the immobilization of thermally permeabilized cells. Cell clustering can be bypassed by immobilizing non-permeabilized cells. This immobilization procedure makes additional permeabilization possible.     

Redirecting cellular metabolism by immobilization of cultured plant cells: A model study with Coffea arabica

Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.