Use of <Emphasis Type="Italic">Pichia fermentans</Emphasis> and <Emphasis Type="Italic">Candida</Emphasis> sp. strains for the biological treatment of stored olive mill wastewater

Of 105 isolates screened for growth on plates containing olive mill wastewater (OMW), five were selected and identified as Pichia fermentans (Y1, Y4) and Candida sp. (Y2, Y11, and Y18). On the basis of their ability to use phenol at 716 mg l⁻¹, strains Y2 (15% reduction) and Y4 (18% reduction) were then used to detoxify stored OMW under various operational conditions. Yeast treatment of OMW increased the pH and, in the best conditions (aeration and no glucose addition), the COD decreased (47%) and phytotoxicity was also decreased (56%) probably due to the changes in the composition of phenolic compounds.     

Strategies for dephenolization of raw olive mill wastewater by means of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks. The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO₄). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42–68% for five cycles).     

Two-stage biological treatment of olive mill wastewater with whey as co-substrate

Olive mill wastewater (OMW) is a highly polluting wastewater, caused by a high organic load and phenol content. These characteristics suggest that it may be suitable for aerobic treatment and anaerobic bacterial digestion. Aerobic treatment coupled with anaerobic bacterial digestion may be economically feasible as the methane produced is a valuable energy source while simultaneously purifying the OMW. In an attempt to improve the overall performance of the process, the addition of a co-substrate such as whey to the aerobic treatment pre-treatment of OMW by the yeast Candida tropicalis was studied.The two-stage system operated satisfactorily up to an organic loading rate (OLR) of 3.0 kg COD L⁻¹ day⁻¹ with a biogas production rate of 1.25 L_biogas L_reactor⁻¹ day⁻¹ and a total COD reduction in excess of 93% (62% COD reduction in aerobic pretreatment and 83% COD reduction in anaerobic digestion). Fifty-four percent of the phenol was biodegraded during the aerobic treatment stage, and biogas with between 68% and 75% methane was produced during anaerobic digestion.     

Effects of olive mill wastewater on soil carbon and nitrogen cycling

This study investigated the cycling of C and N following application of olive mill wastewater (OMW) at various rates (0, 42, 84, and 168 m³/ha). OMW stimulated respiration rate throughout the study period, but an increase in soil organic matter was observed only at the highest rate. Soil phenol content decreased rapidly within 2 weeks following application but neither phenol oxidase and peroxidase activity nor laccase gene copies could explain this response. Soil NH₄ ⁺-N content increased in response to OMW application rate, while an opposite trend observed for NO₃ ^?-N, which attributed to immobilization. This decrease was in accordance with amoA gene copies of archaeal and bacterial ammonia oxidizers in the first days following OMW application. Afterwards, although amoA gene copies and potential nitrification rates recovered to values similar to or higher than those in the non-treated soils, NO₃ ^?-N content did not change among the treatments. A corresponding increase in denitrifying gene copies (nirK, nirS, nosZ) during that period indicates that denitrification, stimulated by OMW application rate, was responsible for this effect; a hypothesis consistent with the decrease in total Kjeldahl nitrogen content late in the season. The findings suggest that land application of OMW is a promising practice for OMW management, even at rates approaching the soil water holding capacity.     

Chemical and toxic evaluation of a biological treatment for olive-oil mill wastewater using commercial microbial formulations

Olive-oil-mill wastewater (OMW) has significant polluting properties due to its high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and phenols. In the present study, different commercial bacterial formulations were used in the biological treatment of OMW. COD and toxicity testing using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna) were employed to evaluate abatement of the organic load and reduction of the toxic potential. In addition, the four most active formulations were tested mixed pair-wise on the basis of their unique characteristics in order to evaluate the improvement of treatment. The effect of treatment was assessed by measuring COD removal, reduction of total phenols, and decreased toxicity. The results obtained with the mixed formulations showed that the maximum removal of the organic load was about 85%, whereas phenols were reduced by about 67%. The toxicity for rotifers decreased by 43% and for crustaceans by about 83%.     

Olive mill wastewater remediation by means of Pleurotus ostreatus

Results of a research program on raw olive-mill wastewater (OMW) bioremediation are presented. Bioremediation experiments have been carried out both in an airlift bioreactor and in aerated flasks, using Pleurotus ostreatus. The process was investigated under controlled non-sterile operating conditions, representative of industrial operation. Growth of P. ostreatus as well as polyphenols conversion were assessed. OMW bioconversion was characterized in terms of total organic carbon, polyphenols concentration, phenol oxidase activity, extent of decolourization and pH as a function of time. Results demonstrate that: P. ostreatus effectively grows on raw OMW; polyphenols abatement is controlled by the availability of nutrients and can be as large as 95%; bioconversion of non-sterilized OMW does not result into appreciable decolourization of the liquid medium. The use of an internal loop airlift bioreactor as a candidate for the full-scale implementation of an OMW aerobic bioremediation process is demonstrated.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

Use of cheese whey to enhance <Emphasis Type="Italic">Geotrichum candidum</Emphasis> biomass production in olive mill wastewater

Geotrichum candidum is a yeast-like filamentous fungus that has attracted industrial interest. The present work investigated G. candidum biomass production in agro-industrial wastewaters (olive mill wastewater (OMW) and cheese whey (CW)) as the only substrate. Different solid media (Sabouraud dextrose agar (SDA), CW, OMW, and OMW/CW mixtures in different proportions) were tested. OMW/CW mixtures proved to be suitable for optimal mycelia growth of G. candidum with a very high hyphae density. The highest fungal and expansion rate growth of 83 ± 1 mm and 12.4 day⁻¹, respectively, were obtained on a 20:80 mixture of OMW/CW, which was incubated for 7 days. This optimal mixture was used to study the biomass production and the OMW decolorization ability of G. candidum in the presence of CW in liquid medium. Liquid cultures were also conducted in OMW and CW separately. After 5 days of incubation, fungal biomass reached 9.26 g l⁻¹ in the OMW/CW mixture and only 2.83 g l⁻¹ in CW, while no biomass production was observed in OMW alone. OMW decolorization and dephenolization by G. candidum also improved in the presence of CW with a decolorization efficiency of 54.5% and a total phenolic reduction of 55.3%, compared with the control which yielded values of about 10% and 15%, respectively. These results suggested that OMW/CW—as the only substrate—could be used as a cost-effective medium to produce G. candidum biomass, without the need for water dilution or supplementation with other nutriments.     

Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

Far‐red dependent changes in the chemical composition of Spirulina platensis

The influence of far‐red light (FRL) was studied on the chemical composition of Spirulina platensis biomass. The following light compositions were used during the culture white light, blue‐red LED light (BRL) and BRL supplemented with FRL (BRFRL). Chlorophyll and phenol contents were measured by spectrophotometric methods, whereas presence of carotenoids, lipids, and phycobiliproteins were estimated by Fourier‐transform Raman spectrometry. Additionally, phenol content was investigated by fluorescence intensity of algae culture in the range of 430–650 nm. The content of chlorophyll and phenols in algae cells depended on the spectral composition of light and was the highest under BRL (16.7 ± 0.5 and 9.1 ± 0.6, respectively). It was shown that there is a positive linear correlation (R = 0.902 at p < 0.0000001) between the ratio of relative fluorescence intensity of S. platensis suspensions at 450 nm to the suspensions at 540 nm (F450/F540) and the content of phenolic compounds in the biomass. Changes in the F450/F540 ratio can explain approximately 80% changes of phenol contents in algae cells. Spirulina platensis Raman spectra demonstrated that the biomass of algae growing under white light and BRL had a significantly higher intensity of phycobiliprotein bands than the algae growing under BRFRL.     

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete chrysosporium

Summary Olive oil mill wastewater (OMW) has a high organic load, and this is a serious concern of the olive industry. Conventional biological wastewater treatments, despite their simplicity and suitable performance are ineffective for OMW treatment since phenolics possess antimicrobial activity. In order to carry out a proper treatment of OMW, use of a microorganism able to degrade the phenolics is thus necessary. In this study the ability of Phanerochaete chrysosporium to degrade the phenolic compounds of OMW and to decrease the chemical oxygen demand (COD) using cells immobilized on loofah was examined. The basal mineral salt solution along with glucose, ammonium sulfate and yeast extract was used to dilute the OMW appropriately. The fungus did not grow on the concentrated OMW. The extent of removal in this bio-treatment, of total phenols (TP) and the COD were 90 and 50%, respectively, while the color and aromaticity decreased by 60 and 95%, respectively. The kinetic behavior of the loofah-immobilized fungus was found to follow the Monod equation. The maximum growth rate μ_max was 0.045 h⁻¹ while the Monod constant based on the consumed TP and COD were (mg/l) 370 and 6900, respectively.     

Decolourization of olive mill waste-waters by the white-rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system

Summary The decolourization of olive mill waste-waters (OMW) by Phanerochaete chrysosporium was investigated. OMW decolourization occurred during the primary phase of growth when glycerol was used as the carbon source, and during secondary metabolism in nitrogen-limited cultures. The decolourization was found to be extensive (74% of colour removal, 80% of chemical oxygen demand removal) when the cultures were supplement d with veratryl alcohol and flushed with O₂. The biodegradation system was repressed with glutamate as a nitrogen source. These results suggest that all or part of the lignin-degrading system of P. chrysosporium played a role in biodegradation of OMW. The decolourization of OMW corresponds to depolymerization of high-molecular-mass aromatics combined with mineralization of a wide range of monoaromatic compounds. Correspondence to: S. Sayadi     

Decolourization of Black Oxidized Olive-Mill Wastewater by a New Tannase-Producing Aspergillus flavus Strain Isolated from Soil

Summary By contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3).     

The use of hydroxyl-radical-generating systems for the treatment of olive mill wastewaters

Three hydroxyl-radical producing biomimetic systems, composed of CuII, hydrogen peroxide and pyridine, glucaric or succinic acid, were able to perform decolorization of olive mill wastewaters (OMW) >85 % within 3 d combined with a significant removal of total phenols (>75 %). The systems consisting of 50 mmol/L succinic acid, 5-10 mmol/L CuSO4 and 100 mmol/L H2O2 were the most effective at OMW treatment, and led to the reduction of phenol contents to <1 % along with high decolorization (>88 %) and acceptable values of chemical oxygen demand.     

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682—C. boidinii and 625, 642, 647, and 941—P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10–30°C) and (NH₄)₂SO₄ (0.0–6.0 g l⁻¹) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10°C with 6.0 g l⁻¹ of (NH₄)₂SO₄ added.     

Xanthan production from olive-mill wastewaters

Olive mill wastewaters (OMW) are a by-product from olive oil manufacture that cause environmental pollution. These wastes have been used as substrate for the production of the extracellular polysaccharide xanthan by Xanthomonas campestris NRRL B1459-S4L41. Growth and xanthan production on dilute OMW as a sole source of nutrients were obtained at OMW concentrations below 60%, yielding a maximal xanthan production of 4.4gl⁻¹ at 30–40% OMW concentration. Addition of nitrogen and/or salts led to significantly increased xanthan yields with a maximum of 7.7gl⁻¹. The N/salts supplements also allowed an increase in the optimal OMW concentration. Inocula pre-grown on OMW can be used. Results suggest that an improved xanthan yield could be obtained with adequate balance between waste concentration and nitrogen or salt supplementation. OMW is proposed as a low-cost substrate for xanthan production with the additional environmental benefit of this use.     

Dephenolisation of olive mill wastewater using adapted Trametes versicolor

Olive mill wastewater (OMW) is an effluent of the olive oil extraction process. The large volumes involved, along with the high phenolic content and chemical oxygen demand, cause major environmental problems. The presence of phenolics limits the effectiveness of aerobic or anaerobic treatment of this wastewater. In most of the studies performed on OMW, the concentration of phenolics is reduced by diluting the OMW prior to biological treatment, which leads to an increase in waste volume. Therefore, the aim of this work was to investigate the possibility of reducing the phenolic content without dilution and without any addition of nutrients or pretreatment by using the white-rot fungi Trametes versicolor FPRL 28A INI. Through an adaptation process, the fungus was able to remove 78% of total phenolics in shake flask experiments and 39% in static culture using undiluted OMW medium. In continuously stirred tank reactor (CSTR) conditions, 70% of total phenolics removal was achieved. Analysis with GC–MS showed that all simple phenolics disappeared from the medium after the 8th day of cultivation at an 0.25 vvm aeration rate. The maximum activities of phenol degrading enzymes laccase and manganese peroxidase (MnP) obtained under these conditions were 762.14 ± 42.11 and 97.80 ± 8.11 U l^?1 respectively.     

New Bromoperoxidase from Coralline Algae that Brominates Phenol Compounds

Bromoperoxidases were found in coralline algae (Corallinaceae, Rhodophyta) collected from seasides in Japan, and high enzyme activities were observed in Corallina officinalis, Corallina pilulifera and Amphiroa zonata. The optimum pHs of the enzymes from coralline algae were around 6.0. The enzymes were specific for I^? and Br^?, and did not act on Cl^? and F^?. The enzymes purified from Corallina pilulifera and Amphiroa ephedraea catalyzed the brominations of phenol and O-hydroxybenzyl alcohol in the presence of Br^? and H₂0₂ to form the same product, 2,4,6-tribromophenol.     

Effect of an anaerobic bacterial consortium isolated from termites on the degradation of olive-mill waste-water

Summary A microbial consortium obtained by enrichment culture on syringate of termite gut material was used to improve the anaerobic degradation of olive-mill waste-water (OMW). Addition of the consortium (1/4 v/v) to the control inoculum originating from waste-water sludge, increased methane production by 50% over the control during anaerobic digestion of OMW prefermented by Aspergillus niger. This increase was related to enhanced acetate production in the presence of the consortium. When OMW was not prefermented by A. niger, no improvement in methane production was observed, indicating that the aerobic degradation of inhibitory substances is needed for the consortium to express its potential. Correspondence to: J. L. Garcia