Diversity and osmoregulatory responses of bacteria isolated from two-phase olive oil extraction waste products

Six phenotypically distinct groups of bacteria were isolated from Spanish and Greek sources of alpeorujo, the waste from two-phase decanter, continuous extraction olive oil mills. These different bacteria isolated from alpeorujo showed different growth and osmoregulatory responses to conditions of reduced water activity (a_w), and there was a correlation between the ability of isolates to withstand low a_w and grow on alpeorujo. One isolate (1A), which grew particularly well both on alpeorujo and in nutrient broth containing either 10% NaCl or 30% sucrose, was identified as being most closely related to Bacillus amyloliquifaciens using biochemical tests and partial 16S rDNA gene sequence analysis. Bacillus sp. strain 1A was found to display an atypical membrane lipid response at low a_w since the major change was an increase in the zwitterionic phosphatidylethanolamine rather than an anionic phospholipid such as phosphatidylglycerol. In addition, instead of the expected decrease, there was an increase in the average lipid fatty acid acyl chain length at low a_w without any other compensatory fluidizing change. This revised version was published online in July 2006 with corrections to the Cover Date.     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Yeast screening from avermectins wastewater and investigation on the ability of its fermentation

Wastewater from avermectin production is refractory with high output, high chemical oxygen demand (COD) concentration and high cost further. The current wastewater treatment technology, with little reuse of the organic material, needs high dilution ratio during treatment. Yeast single cell protein was produced from the wastewater of avermectins fermentation in this research. First, the yeast strain (H-1) which show enough tolerance to avermectins residue was screened from the wastewater, and it was identified as being most closely related to Candida tropicalis (100%, EF120592.1) using 18S rDNA gene sequence analysis. Second, its growth characteristics in the avermectins wastewater were studied. The dry biomass reached the maximal point of 10 g/L, the COD removal was up to 66.67%, and avermectins removal in the wastewater was 99.48% at the optimal condition that it was liquid volume 50 ml (250 ml flask), pH 4.0, temperature 30°C, inoculum volume 10% (V/V) and fermentation period 20 h. Third, the nutritional contents of dry yeast powder were determined, in which the water content was 8.12%, ash content was 5.18% and the crude protein was 40.02%. The dry yeast powder from avermectin waste liquor was promising to be used as a raw material or nitrogen source for commercial production of avermectins. The project was economically feasible by primary cost accounting.     

Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors

Two-phase olive mill waste (TPOMW) is a semi-solid effluent that is rich in contaminating polyphenols and is produced in large amounts by the industry of olive oil production. Laboratory-scale bioreactors were used to investigate the biodegradation of TPOMW by its indigenous microbiota. The effect of nutrient addition (inorganic N and P) and aeration of the bioreactors was studied. Microbial changes were investigated by PCR-temperature time gradient electrophoresis (TTGE) and following the dynamics of polar lipid fatty acids (PLFA). The greatest decrease in the polyphenolic and organic matter contents of bioreactors was concomitant with an increase in the PLFA fungal/bacterial ratio. Amplicon sequences of nuclear ribosomal internal transcribed spacer region (ITS) and16S rDNA allowed identification of fungal and bacterial types, respectively, by comparative DNA sequence analyses. Predominant fungi identified included members of the genera Penicillium, Candida, Geotrichum, Pichia, Cladosporium, and Aschochyta. A total of 14 bacterial genera were detected, with a dominance of organisms that have previously been associated with plant material. Overall, this work highlights that indigenous microbiota within the bioreactors through stimulation of the fungal fraction, is able to degrade the polyphenolic content without the inoculation of specific microorganisms.     

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.     

Potential of agroindustrial waste from olive oil industry for fuel ethanol production

Olive pulp (OP) is a highly polluting semi-solid residue generated from the two-stage extraction processing of olives and is a major environmental issue in Southern Europe, where 80% of the world olive oil is produced. At present, OP is either discarded to the environment or combusted with low calorific value. In this work, utilization of OP as a potential substrate for production of bioethanol was studied. Enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for OP from 10% to 30% dry matter (i.e., undiluted). Enzymatic hydrolysis resulted in an increase in glucose concentration by 75%, giving final glucose yields near 70%. Fermentation of undiluted OP hydrolysate (OPH) resulted in the maximum ethanol produced (11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar for all tested OPH concentrations and were in the range of 0.49-0.51 g/g. Results showed that yeast could effectively ferment OPH even without nutrient addition, revealing the tolerance of yeast to OP toxicity. Because of low xylan (12.4%) and glucan (16%) content in OP, this specific type of OP is not a suitable material for producing only ethanol and thus, bioethanol production should be integrated with production of other value-added products.     

Biotechnological conversion of waste cooking olive oil into lipid-rich biomass using Aspergillus and Penicillium strains

Aims: In this study, we have investigated the biochemical behaviour of Aspergillus sp. (five strains) and Penicillium expansum (one strain) fungi cultivated on waste cooking olive oil. The production of lipid‐rich biomass was the main target of the work. In parallel, the biosynthesis of other extracellular metabolites (organic acids) and enzyme (lipase) and the substrate fatty acid specificity of the strains were studied. Methods and Results: Carbon‐limited cultures were performed on waste oil, added in the growth medium at 15 g l^?1, and high biomass quantities were produced (up to c. 18 g l^?1, conversion yield of c. 1·0 g of dry biomass formed per g of fat consumed or higher). Cellular lipids were accumulated in notable quantities in almost all cultures. Aspergillus sp. ATHUM 3482 accumulated lipid up to 64·0% (w/w) in dry fungal mass. In parallel, extracellular lipase activity was quantified, and it was revealed to be strain and fermentation time dependent, with a maximum quantity of 645 U ml^?1 being obtained by Aspergillus niger NRRL 363. Storage lipid content significantly decreased at the stationary growth phase. Some differences in the fatty acid composition of both cellular and residual lipids when compared with the initial substrate fat used were observed; in various cases, cellular lipids more saturated and enriched with arachidic acid were produced. Aspergillus strains produced oxalic acid up to 5·0 g l^?1. Conclusions: Aspergillus and Penicillium strains are able to convert waste cooking olive oil into high‐added‐value products. Significance and Impact of the Study: Increasing fatty wastes amounts are annually produced. The current study provided an alternative way of biovalourization of these materials, by using them as substrates, to produce added‐value compounds.     

片烟产果胶酶细菌的鉴定及酶活测定

以果胶为碳源, 对津巴布韦片烟烟叶表面产果胶酶细菌进行分离, 采用16S rDNA限制性酶切片段长度多态性分析(ARDRA)和测序方法, 结合形态学、生理生化实验, 对所分离产果胶酶菌株进行鉴定, 同时研究培养时间、温度、起始pH、接种量对菌株产酶的影响。结果表明, 从津巴布韦片烟烟叶表面分离得到的产果胶酶菌株主要为芽孢杆菌属的枯草芽孢杆菌Bacillus subtilis和产碱菌属的粪产碱菌Alcaligenes faecalis。在所分离的菌株中, 枯草芽孢杆菌T10酶活力最高, 以6%的接种量, 在温度为35 °C、起始pH为7.5条件下培养48?56 h, 其果胶酶酶活为571 U/mg, 聚半乳糖醛酸裂解酶酶活为297 U/mg。     

Identification of a newly isolated erythritol-producing yeast and cloning of its erythritol reductase genes

A new erythritol-producing yeast (strain BH010) was isolated in this study. Analysis of the D1/D2 domain of the 26S rDNA sequence, the ITS/5.8S rDNA sequence, and the 18S rDNA sequence allowed the taxonomic position of strain BH010 to be discussed and it was identified and named Moniliella sp. BH010. Physiological characteristics were described. Scanning electron micrography clearly indicated that the cells were cylindrical to elliptical with an average size of 5?×?10?μm when growing in liquid medium, and that pseudohyphae and blastoconidia were observed when cultivated in agar plates. The erythritol reductase genes were cloned, sequenced, and analyzed. BLAST analysis and multiple sequence alignment demonstrated that erythritol reductase genes of Moniliella sp. BH010 shared very high homology with that of Trichosporonoides megachiliensis SNG-42 except for the presence of introns. The deduced amino acid sequences showed high homology to the aldo–keto reductase superfamily.     

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.     

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Physiological and morphological characteristics of yeasts isolated from waste oil effluents

Pichia membranaefaciens, Cryptococcus laurentii, Rhodotorula glutinis and Candida krusei were isolated from contaminated sites. A significant variability in cell forms and in assimilation profiles was observed in the C. krusei strains. The chitin synthase activity and chitin content allowed us to differentiate three strain types. The variability of the phenotypic traits was higher in C. krusei strains isolated from heavily polluted sites. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.     

高产葡萄糖耐量因子酵母的筛选及其发酵特性的研究

通过将供试菌株接种于梯度浓度含铬YEPD平板和液体YPD培养基中进行固体培养及液体发酵试验,利用氨水提取GTF和火焰原子吸收光谱法检测其有机铬含量,从本试验室收藏7株酵母菌种和7株经航天诱变育种的酵母菌种中筛选得到一株葡萄糖耐量因子的高产酵母,有行机铬含量达1287ug/g,总铬含量达1917ug/g.同时研究了菌体发酵过程中,菌体有机铬富集量和其发酵参数的动态关系.     

Vitamin and amino acid requirements of Lactobacillus plantarum strains isolated from green olive fermentations

The requirement for essential amino acids and vitamins was determined in wild-type Lactobacillus plantarum strains isolated from green olive fermentation brines. All the strains were found to be auxotrophic with respect to the amino acids but some of them were prototrophic for pyridoxal, p -aminobenzoic acid and/or nicotinic acid. Their growth response to these nutrients was also studied and found to be quite heterogeneous. Nutritional requirement pattern as a criteria for selecting starter cultures is discussed.     

Effects of a compost made from the solid by-product ("alperujo") of the two-phase centrifugation system for olive oil extraction and cotton gin waste on growth and nutrient content of ryegrass (Lolium perenne L.)

A pot experiment was conducted on a low-fertility calcareous soil in order to evaluate the effect on ryegrass growth and nutrient uptake of an organic fertiliser obtained by composting "alperujo" and cotton gin waste. Compost, alone and combined with nitrogen fertilisation, was added to the soil at three rates and three harvests were obtained. The compost application enhanced plant growth in the first and third harvest. However, the additional nitrogen fertilisation clearly improved soil productivity due to the scarce availability of this nutrient in the compost. Also, a general increase in the plant contents of phosphorus and potassium in the first two harvests was recorded, whereas treatments with the maximum compost rate showed the highest plant content of copper in the last two harvests. Decreases in calcium in the last two harvests, in magnesium in all of them and in iron and manganese in the last harvest were also observed.     

Lipolysis of olive oil and tallow in an emulsifier-free two-phase system by the lipase from oat seeds (Avena sativa L.)

Summary Lipase activity from whole oat seeds is dependent upon calcium ion in a two phase olive oil-water system. No other tested ion can substitute for calcium. The lipolysis of melted tallow at 46°C was successfully conducted in the presence of calcium ion; the fatty acid fraction contained predominantly oleic acid.