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     

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Summary Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically ¹⁴C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.     

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.     

Covalent coupling of microorganisms to a cellulosic support

Methods for the covalent coupling of microorganisms to a solid support were investigated. Both bacteria and yeast were attached to cellulose particles using cyanuric chloride as the coupling agent, although different experimental procedures were needed for the two types of microbes. This general technique for whole-cell immobilization offers an advantage over entrapment methods in that the cells are attached to the outer surface of the solid, thus eliminating the resistance of a gel to the transfer of nutrients and products. There are also indications that such immobilized cells show high productivities.     

Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor

Production of fuel ethanol from sugar beet juice, using cells immobilized on loofa sponge was investigated. Based on ethanol productivity and ease of cell immobilization, a flocculating yeast strain, Saccharomyces cerevisiae IR2 was selected for ethanol production from sugar beet juice. It was found that raw sugar beet juice was an optimal substrate for ethanol production, requiring neither pH adjustment nor nitrogen source supplement. When compared with a 2 l bubble column bioreactor, mixing was not sufficient in an 8 l bioreactor containing a bed of sliced loofa sponges and consequently, the immobilized cells were not uniformly distributed within the bed. Most of the cells were immobilized in the lower part of the bed and this resulted in decreased ethanol productivity. By using an external loop bioreactor, constructing the fixed bed with cylindrical loofa sponges, dividing the bed into upper, middle and lower sections with approximately 1 cm spaces between them and circulating the broth through the loop during the immobilization, uniform cell distribution within the bed was achieved. Using this method, the system was scaled up to 50 l and when compared with the 2 l bubble column bioreactor, there were no significant differences (P > 0.05) in ethanol productivity and yield. By using external loop bioreactor to immobilize the cells uniformly on the loofa sponge beds, efficient large scale ethanol production systems can be constructed.     

Enhanced epsilon-poly-L-lysine production from Streptomyces ahygroscopicus by a combination of cell immobilization and in situ adsorption

Epsilon-poly-L-lysine (epsilon-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce epsilon-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more epsilon-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced 0.54 +/- 0.1 g/l epsilon-PL, which significantly increased to 3.64 +/- 0.32 g/l after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded 2.73 +/- 0.26 g/l of epsilon-PL, an increase from 0.82 +/- 0.08 g/l. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an epsilon-PL total amount of 8.05 +/- 0.84 g/l was obtained. The proposed combination method offers promising perspectives for epsilon-PL production.     

Bacterial cellulose–alginate composite sponge as a yeast cell carrier for ethanol production

A bacterial cellulose–alginate (BCA) sponge, fabricated by a freeze-drying process, was successfully used as a yeast cell carrier for ethanol fermentation. The BCA sponge exhibited several advantageous properties, such as high porosity, appropriate pore size, strong hydrophilicity and high mechanical, chemical and thermal stabilities. BCA has an asymmetric structure, with a thin, dense outer layer covering an interior of interconnected macropores that are distributed throughout the sponge, which is effective for yeast immobilization. At 48 h of the fermentation, the maximum ethanol concentration produced by the immobilized culture (IC) in the BCA carrier was about 100 g/L, which was approximately 13% and 45% higher than that from the suspended culture (SC) and from IC in Ca-alginate matrix, respectively. Repeated-batch ethanol productions using IC in BCA carriers were also more stable than those using SC or IC in Ca-alginate matrix. The results of a 15 cycle repeated batch operation demonstrated that the system with IC in BCA exhibited superior long-term stability for ethanol fermentation with the average ethanol productivity at 1.9 g/L h and the immobilized yield at 86%. The improved ethanol fermentation performance was mainly due to the water uptake ability and properly interconnected pore structure, which help to overcome limiting mass transfer.     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Development of a method for immobilization of non-flocculating cells in loofa (Luffa cylindrica) sponge

Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture.     

Cyclodextrin glycosyltransferase production by free cells of <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R in batch fermentation and by immobilized cells in a semi-continuous process

Cyclodextrin glycosyltransferase (CGTase) catalyzes starch conversion into cyclic or linear oligosaccharides, important industrial products for the complexation of non-polar substances. In this work, conditions to increase CGTase production from Bacillus circulans strain DF 9R were optimized by two systems. On one hand, free cells were grown in batch fermentation experiments to optimize aeration and pH. The highest activity (1.47 ± 0.21 U ml^?1) was achieved after 48 h of growth, aeration of 1.5 vvm and pH regulated to 7.6. On the other hand, bacterial cells were immobilized on loofa and synthetic sponge, and used for CGTase production in a semi-continuous process. An initial biomass of 30 mg of lyophilized cells and an immobilization time of 24 h with loofa or synthetic sponge were enough to achieve increased production of CGTase: 0.91 ± 0.10 and 0.95 ± 0.11 U ml^?1, respectively. Sponges with immobilized bacteria were reused in 12 successive cycles. Besides, in our conditions, CGTase was not adsorbed onto the supports used for immobilization, which ensured the total recovery of the enzyme from the culture medium. The two CGTase production processes studied showed similar productivity and could be potentially scaled up.     

Immobilization of thylakoids in porous particles and stabilization of the photochemical processes by glutaraldehyde action at subzero temperature

Summary Lettuce thylakoids were immobilized by the action of glutaraldehyde at sub-zero temperature in presence of albumin. Particles with sponge structure and good mechanical properties were obtained. The activity yield after immobilization was found equal to 70% for the oxygen production. Photosystems I and II were both active after immobilization. The yield for the ATP production was 27%. An increase of stability of the immobilized thylakoids was observed when stored under illumination.     

Bioactivity of immobilized microalgal cells: application potential of vegetable sponge in microbial biotechnology

Bioactivity of immobilized cells, as evidenced by the production of intracellular phycoerythrin and extracellular polysaccharides by Porphyridium cruentum , in batch and repeated batch culture, respectively, is reported. Cells of P. cruentum immobilized within the vegetable sponge of Luffa cylindrica were grown in batch culture for 24 d and maintained at the stationary phase of growth in repeated batch cultures in a growth-limiting medium for 50 d. The study indicates the application potential of this recently reported biostructure as an immobilization matrix in microbial biotechnology.     

Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria

Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains. The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polarity in filamentous fungi: moving beyond the yeast paradigm

Filamentous fungi grow by the polar extension of hyphae. This polar growth requires the specification of sites of germ tube or branch emergence, followed by the recruitment of the morphogenetic machinery to those sites for localized cell wall deposition. Researchers attempting to understand hyphal morphogenesis have relied upon the powerful paradigm of bud emergence in the yeast Saccharomyces cerevisiae. The yeast paradigm has provided a useful framework, however several features of hyphal morphogenesis, such as the ability to maintain multiple axes of polarity and an extremely rapid extension rate, cannot be explained by simple extrapolation from yeast models. We discuss recent polarity research from filamentous fungi focusing on the position of germ tube emergence, the relaying of positional information via RhoGTPase modules, and the recruitment of morphogenetic machinery components including cytoskeleton, polarisome and ARP2/3 complexes, and the vesicle trafficking system.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

微贮用植物乳杆菌固定化菌剂的制备

乳酸菌与纤维素降解菌因其可防止微贮饲料酸败、增加秸秆饲料的营养价值等优点,在秸秆微贮过程中起重要作用。但由于乳酸菌的繁殖会抑制纤维素降解菌的活性,如何实现微贮过程中两种微生物分时发挥功能是解决上述问题的关键。文中利用固定化技术将乳酸菌制备成含有玉米秸秆粉的固定化菌剂以达到缓释的目的。首先制作固定化空白小球得出复合固定化载体成球的最佳浓度,利用玉米芯吸附植物乳杆菌S1得到复合固定化载体,以对S1的包埋率、成球效果等为指标,通过对比两种固定化方法 (包埋法与包埋-交联法),得到固定化植物乳杆菌S1的最佳条件。研究表明,使用6%PVA+0.4%SA+0.3%CMC-Na进行包埋-交联时成球效果最好,使用1.2%SA+0.5%CMC-Na进行直接包埋时成球效果最好。通过对比5种固定化工艺,将1.2%SA+0.5%CMC-Na和吸附玉米粉组成的固定化载体混合物逐滴滴入4%氯化钙中直接包埋24 h得到的固定化小球其机械强度以及包埋率均优于其他工艺。因此,利用玉米芯吸附-海藻酸钠包埋的方法可以有效提高植物乳杆菌包埋效率,为使用固定化技术制备微贮饲料菌剂奠定基础。     

Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.     

壳聚糖球固定化酵母细胞的研究

以戊二醛为交联剂,将壳聚糖球交联引入醛基,然后将交联的壳聚糖球浸泡在酵母细胞悬浮液中,制备了固定化酵母细胞壳聚糖球。以苯乙酮酸为底物,催化合成了D-扁桃酸。最优固定化条件是戊二醛的质量分数w(GA)=1%,酵母细胞与交联壳聚糖球的质量比m(Y):m(CB)0=0.5,交联时间为6h,固定化时间为18h,底物浓度为10mmol/L,在此条件下反应最大转化率和产物光学纯度分别高达67.86%和98.05?。固定化酵母壳聚糖球具有良好的重复使用性和贮存稳定性。     

Oxidation of hydrogen sulfide by flocculated Thiobaccillus denitrificans in a continuous culture

In a continuous fermentation, significant advantages may be gained by immobilization of microbial cells. Immobilization allows cells to be retained in the fermenter or to be readily recovered and recycled. Therefore, the hydraulic retention time and the biomass retention time are decoupled. A novel cell immobilization has been developed for the immobilization of autotrophic bacteria by coculture with floc-forming heterotrophic bacteria with growth of the latter limited by the availability of organic carbon. The result is an immobilization matrix which grows along with the immobilized autotroph. We have previously demonstrated the utility of this approach by immobilizing the chemoautotroph Thiobacillus denitrificans in macroscopic floc by coculture with floc-forming heterotrophs from an activated sludge treatment facility. Floc with excellent settling characteristics were produced. These floc have now been used to remove H(2)S from a gas stream bubbled through continuous cultures. The stoichiometry and kinetics of H(2)S oxidation by immobilized T. denitrificans were comparable to that reported previously for free-cell cultures. Oxygen uptake measurements indicated the growth of both T. denitrificans and the heterotrophs although the medium contained no added organic carbon. Continuous cultures with total biomass recycle were maintained for up to four months indicating the long-term stability of the commensal relationship between the immobilized autotroph and the heterotrophs which composed the immobilization matrix. It was observed that at any given H(2)S loading the biomass concentration reached a maximum and leveled out. The ultimate biomass concentration was dependent upon the H(2)S feed rate.