Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

Production of a Bioflocculant by Rhodococcus erythropolis S-1 Grown on Alcohols

Rhodococcus erythropolis strain S-1, which was isolated from soil, produces a bioflocculant. We have found that alcohols are useful carbon sources for its flocculant production. Ethanol was best for flocculant production and culture time. The bioflocculant produced on ethanol medium flocculated a wide range of suspended soils, alkaline and acid.     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Conditions for Production of Microbial Cell Flocculant by Aspergillus sojae AJ7002

Cultivation is reported on Aspergillus sojae AJ7002 which synthesized an extracellular bio-flocculant. Growth studies in shaking flasks and fermentors were conducted to obtain higher flocculant production. The highest level of polymer accumulation was attained after 48–72 hr cultivation at 30–34°C. The favorable substrates for polymer formation were casein, yeast extract, polypepton and amino acids, such as glutamic acid and alanine. The addition of saccharides to the medium was found to reduce the pH of the culture broths, and hence inhibit the accumulation of flocculant in the culture broth. The finding that the product was a single substance from the early stage of fermentation suggested that the polymer was not a product of cell autolysis. The components of the polymer which were produced by Asp. sojae did not vary even if the medium composition or culture condition changed. The addition of 2-ketogluconic acid, which is one of the constituents of the polymer increased the flocculating activity of the culture medium.     

Optimization of Chlorella biomass harvesting by flocculation and its potential for biofuel production

Optimization of microalgal biomass harvesting is essential to produce effective and optimum outcomes that can contribute towards a feasible and economical harvesting technique. Two Chlorella species were used, namely, C. vulgaris and C. sorokiniana UKM3. Two essential factors affecting microalgal biomass harvesting via flocculation, namely, the initial pH of the microalgal broth and flocculant (chitosan) concentration were studied. The optimization process was conducted by using a response surface methodology (RSM) based on the model of face-centered-central composite design (FC-CCD). The potential for biofuel application of the harvested biomass was evaluated based on the production of fatty acid methyl esters (FAMEs) by transesterification. The quadratic models obtained from the RSM significantly fitted the experiment data as the p-values were less than 0.05. The initial pH of the microalgal suspension was found to have a more significant effect on the flocculation process than flocculant concentration. For C. vulgaris, the highest flocculant efficiency of 98.7% was obtained at a chitosan concentration of 0.2 g L^?1 and an initial pH of 12.0, whereas for C. sorokiniana UKM3, at 0.15 g L^?1 of chitosan and initial pH of 12.0 produced the highest efficiency of 97.1%. The harvested biomass of both species exhibited a high content of palmitic acid (C16:0) with 29.74 wt% and 11.81 wt% of dry biomass for C. vulgaris and C. sorokiniana UKM3, respectively. This study showed that Chlorella species can be harvested efficiently using the flocculation process and manifested an excellent potential for biodiesel production where palmitic acid (C16:0) is one of the main compounds for high-acid oil-biodiesel.

     

Screening of flocculant-producing microorganisms and some characteristics of flocculants

Summary 27 strains of microorganisms including moulds, bacteria, actinomyces and yeasts, which produced substances that flocculated a kaolin clay, were screened. One strain, identified as Arthrobacter sp., secreted bioflocculant during growth. The flocculant (MF-6) was purified to electrophoretic homogeneity by precipitation with ethanol and gel chromatography. Its molecular weight was higher than 10⁶ Da. It could efficiently flocculate all suspended solids tested in aqueous solution and colored water like dye solution, and its flocculating ability was significantly enhanced by adding Ca²⁺, Mn²⁺, Mg²⁺, etc.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Production of <Emphasis Type="Italic">Anti-Helicobacter pylori</Emphasis> metabolite by the lichen-Forming fungus <Emphasis Type="Italic">Nephromopsis pallescens</Emphasis>

The present study was conducted to evaluate the antibacterial activity of lichen-forming fungi (LFF) against Helicobacter pylori, and to optimize the culture conditions of LFF for maximum production of natural antibiotics against H. pylori. To accomplish this, a screening assay was first conducted among 19 species of LFF. The extract of Nephromopsis pallescens (KOLRI-040516) exhibited the strongest anti-ff. pylori activity. Bioautograghic TLC and HPLC analysis identified usnic acid as the main antibacterial substance produced by JV. pallescens. The growth of JV. pallescens and production of antibacterial substances produced by the fungus were then investigated under several culture conditions including the culture media, initial medium pHs, incubation temperatures, and the degree of aeration. The results indicated that culture in MY medium with an initial pH of 6.0, a temperature of 15°C and a low degree of aeration supported the largest usnic acid production of the fungus (16.4 ug usnic acid/g dry biomass). Especially, aeration was found to be an important factor that affect both growth and usnic acid production of N. pallescens.     

Production of taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosa-sinensis

Taxol is a highly functionalized anticancer drug widely used in hospitals and clinics. The leaf spot fungus, Phyllosticta dioscoreae was isolated from diseased leaves of Hibiscus rosa-sinensis and screened for extracellular production of taxol in M1D (Modified liquid medium) and PDB (Potato dextrose broth) medium for the first time. The fungus was identified by its morphological and conidial features in the culture growth. The presence of taxol in the fungal culture filtrate was confirmed by different spectroscopic and chromatographic analyses. The amount of taxol produced was quantified by HPLC. The maximum amount of taxol produced was found to be 298 μg/L in M1D medium. Production rate was 5.96 × 10³ times faster than that found in culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol also showed strong cytotoxic activity in vitro in the cultures of human cancer cells tested by apoptotic assay. The results indicate that P. dioscoreae is an excellent source of taxol production, which suggests that the fungus has potential to undergo genetic engineering in order to improve its production level.     

Must deacidification with an induced flocculant yeast strain of Schizosaccharomyces pombe

The use of flocculant cells of the yeast strain Schizosaccharomyces pombe for the deacidification of grape musts in continuous culture was developed. An external loop reactor was used to induce flocculation. The flocs obtained were stable in the pH range 3.0–6.0 and in the presence of several sugars. Some inhibition was observed for high (above 6.0) and low (below 3.0) pH values. Once induced, flocculation could no longer be completely inhibited. Vinho Verde, a typical Portuguese wine, has a relatively low ethanol content and a high acid concentration. The external loop reactor loaded with the flocculant cells was used to deacidify a synthetic medium with sugar and malic acid concentrations similar to the ones found in Vinho Verde grape must. A desirable malic acid decrease with moderate glucose consumption was obtained at a dilution rate of 0.7 H^–1. Improved results were obtained when the synthetic medium was replaced by Vinho Verde grape must. Correspondence to: M. Mota     

A novel flocculant biopolymer produced by Pestalotiopsis sp. KCTC 8637P

Summary A white rot fungus was isolated from rotted leaves and identified as Pestalotiopsis sp. KCTC 8637P. It produced a flocculant biopolymer. A flocculant was partially purified from the culture broth by series of precipitations with 95% ethanol and named as Pestan. The components of Pestan were consisted of glucose : glucosamine : glucuronic acid : rhamnose with a approximately molar ratio of 100:3.5:1.6:1.3.In kaolin suspension(final concentration was 4,800 mg/l), the highest flocculating activity was attained at the biopolymer flocculant concentration of 1 mg/l . The flocculating activity was observed most highest by the addition of cationic solutions, especially 8mM CaCl₂ · 2H₂O or 8mM FeCl₃. The thermal stability of Pestan was sustained up to 70 °C.     

Characterization of a bioflocculant produced by the marine myxobacterium Nannocystis sp. NU-2

The marine myxobacterium strain NU-2, which can grow on high concentrations (up to 7%) of NaCl, was isolated from a salt soil sample collected from the coast of the Huanghai Sea, China. Morphological properties and 16S rDNA sequence analysis indicated that the isolate is a novel species related to the genus Nannocystis. Nannocystis sp. NU-2 produced a new kind of flocculating substance in a starch medium with a yield of 14.8 g l^–1. The NU-2 flocculant was composed of 40.3% proteins and 56.5% polysaccharides, of which glucose, mannose and glucuronic acid were the principal constituents in the relative proportions of 5:4:1. The flocculation activity of the NU-2 flocculant depends strongly on cations such as Fe³⁺ and Al³⁺. When a 30 mg l^–1 FeCl₃ solution is present in kaolin clay suspension, 30 mg l^–1of the flocculant produced a high flocculating activity value of 90%, which remained unchanged over an extensive pH range (pH 2.0–13.0). The flocculant was tested for its ability to bleach dyeing liquors, and the bleaching activities were 98.2% for acid red in 100 mg l^–1of the flocculant and 99.0% for direct emerald blue in 50 mg l^–1of the flocculant under test conditions. Use of the flocculant to bleach basic pink and cation emerald blue liquors was not effective. Electronic Publication     

Purification and Chemical Properties of a Flocculant Produced by Paecilomyces

A new flocculant for microbial cells was purified by ethanol precipitation and gel chromatography from the culture fluid of Paecilomyces sp. I-1. Isoelectric focusing of the flocculant (PF-101) showed a single band at pH 8.5, and its molecular weight was estimated to be over 300,000 daltons by the ultra-filtration method. The results of elemental analysis, the IR spectrum and investigation of the acid hydrolysate by gas and liquid chromatography and colorimetrie analysis suggested that PF-101 was a polysaccharide composed of galactosamine. About 80% of the galactosamine residues were N-unsubstituted and 8% were N-acetylated. Studies on deaminative cleavage, periodate oxidation and Smith degradation suggested that the galactosamine residues were mainly linked by α (→4)-linkaees.     

高效絮凝剂产生菌的筛选及絮凝活性研究

从污泥中分离筛选出1株具有高絮凝活性的细菌菌株F78,对其进行了分子鉴定,测得该菌16SrDNA长度为1501 bp,在GenBank中的登录号为EU443097,序列比对结果显示,该菌与粪产碱杆菌(Alcaligenes faecalis)的16S rDNA碱基序列相似性为99%。絮凝活性物质可由F78菌株分泌到胞外,通过对絮凝活性影响因素的研究,发现Ca2 的助凝效果比Fe3 、Fe2 、K 、Na 好;在高岭土悬液pH 3.0~12.0范围内,能保持较高的絮凝活性;菌株F78产生的絮凝活性物质具有良好的热稳定性。     

Selection of optimal flocculant for effective harvesting of the fucoxanthin-rich marine microalga <Emphasis Type="Italic">Isochrysis galbana</Emphasis>

Flocculation harvesting of the fucoxanthin-rich marine microalga Isochrysis galbana has received little attention. Therefore, we attempted to screen for an optimal chemical flocculant and optimize flocculation conditions from five chemical flocculants—ferric chloride (FC), aluminum sulfate (AS), polyaluminum chloride (PAC), aluminum potassium sulfate (APS), and zinc sulfate (ZS)—for effective flocculation of I. galbana. The growth rate, photosynthetic performance, and fucoxanthin content were determined in re-suspended flocculated algal cells and in the flocculation supernatant cultured algal cells. The results showed that high growth rate and fucoxanthin accumulation were observed when FC was used as the flocculant in I. galbana cultures, which indicated that FC may cause less harm to I. galbana than the other aluminum-based flocculants. Furthermore, satisfactory flocculation efficiency was also observed when FC was used to flocculate I. galbana, and the FC dosage was less than that required for flocculation of I. galbana using PAC, APS, and AS. Thus, we selected FC as the optimal flocculant for harvesting I. galbana based on its flocculation efficiency together with algal physiological performance, growth rate, and fucoxanthin content.     

Screening,Isolation, and Some Properties of Microbial Cell Flocculants

Nineteen out of 214 strains of microorganisms including moulds, bacteria, actinomycetes and yeasts, which were selected from the culture collection of our laboratory were found to produce the substances which flocculate Saccharomyces cerevisiae AJ4005 (baker’s yeast). Among them are Aspergillus sojae, Anixiella reticulata, Geotrichum candidum, Eupenicillium crustaceus, Circinella sydowi, Monascus anka, Sordaria fimicola, Pseudomonas fluorescens, Staphylococcus aureus, Corynebacterium brevicale, Brevibacterium insectiphilum, Streptomyces vinaceus.

The flocculants in the culture broth of these microorganisms precipitated readily by addition of acetone, and flocculated various microorganisms nonspecifically. The flocculant produced by Asp. sojae AJ7002 sedimented activated sludge well.     

Effect of Aeration on Gliotoxin Production by Spergillus Fumigatus in its Culture Filtrate

Gliotoxin, one of the mycotoxins produced by Aspergillus fumigatus, has various, potent bioactivities. However, it has not been considered to be a toxic (or virulence) factor because of its slow production. The aim of the present study was to investigate the effects of aeration on the cytotoxicity of A. fumigatus culture filtrate, and to determine the optimal condition for the rapid production of gliotoxin from this fungus. Fungal culture filtrates were made in three different containers under various conditions of aeration and O₂ concentration. These filtrates were compared in terms of their cytotoxicity on murine macrophages and analyzed by gas chromatography. The culture filtrate showed high cytotoxicity when it was made under highly aerated conditions, but it was significantly less cytotoxic when prepared under non-aerated conditions. The cytotoxic activity became evident within 15 h of culture at 20% O₂, when the fungus had already started producing gliotoxin. The culture filtrates also contained some other as yet unidentified substances that might also to some extent contribute to the cytotoxicity. In light of these results, the authors propose that a highly aerated condition is responsible for the rapid production of gliotoxin, and that gliotoxin might play an important role in the respiratory infection by A. fumigatus, with other toxic substances acting additively or synergistically.     

Investigation on the Toxin Production by Several Blast Fungus Strains and Isolation of Tenuazonic Acid as a Novel Toxin

A survey of the toxin production by several strains of blast fungus was carried out. Among the strains tested only one strain, C₁(THU 69-03) produced α-picolinic acid with good yield and other strains produced neither α-picolinic acid nor piricularin. On the other hand, tenuazonic acid and its Fe-cbelate compound were isolated as novel blast fungus toxins from the surface culture brothes of these strains, but they could not be isolated from the shaking culture brothes. The phytotoxic effect of tenuazonic acid on rice plants in regard to varietal difference was also studied.     

Production of Pectinase and Cellulase by Cladosporium cucumerinum with Dissolved Carbohydrates and Isolated Cell Walls of Cucumber as Carbon Sources

The scab fungus Cladosporium cucumerinum can use pectins and polygalacturonic acid as sole sources of carbon. Cellulose and Ca-polygalacturonate are not available carbon sources for the fungus. When growing on sucrose or pectin, pectinase is produced. In these cases the production of cellulase is insignificant. On a mixture of pectin and carboxymethylcellulose also cellulase is produced. Both pectinase and cellulase are released into the culture filtrate when the fungus grows on cell walls without ionic proteins, whereas only cellulase is released when cell walls with ionic proteins are the carbon source. Pectinase produced by the pathogen can bind to isolated cell walls. The bound pectinase can be extracted with 1 M NaCl from cell walls without ionic proteins, but not from cell walls with ionic proteins. A water-extract or 1 M NaCl-extract of cucumber hypocotyls with visible disease symptoms contains cellulase but no pectinase activity. Lack of pectinase activity in the 1 M NaCl-extract may be due to inhibition by a component that could be extracted by NaCl from the cucumber cell walls.     

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti‐inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized‐cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre‐inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g^?1 and 0.16 mg g^?1 respectively).