Biodegradation of different molecular-mass polyphenols derived from olive mill wastewaters by Geotrichum candidum

The decolourisation of fresh and stored olive mill wastewaters (OMW) and the biodegradation of three groups (F1, F2 and F3) of phenolic compounds by Geotrichum candidum were investigated. Separated phenolic compounds derived from natural OMW ultrafiltration using membranes with a cutoff 2and 100 kDa. G. candidum growth on fresh OMW decreased pH and reduced COD and colour of 75% and 65%, respectively. However, on the stored-black OMW a failure of COD and colour removal were observed. G. candidum activity on this later substrate was enhanced by the addition of a carbon source easily metabolised, misleading an improvement of the COD reduction and decolourization that reached 58% and 48%, respectively. Growth of G. candidum in the presence of F2 or F3 polyphenolic fractions induced high decolourisation and depolymerisation of phenolic compounds. Whereas, very week decolourisation and biodegradation were observed with F1 fraction. Moreover, the highest levels of lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were obtained in the presence of F2 fraction. These results showed that increasing of molecular-mass of aromatics led to an increase in levels of depolymerisation, decolourisation and COD removal by G. candidum culture.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Scale down and optimization of olive mill wastewaters decolorization by Geotrichum candidum

Microbiol flora acclimated in continuous pilot scale bubble column fed with OMW was analysed. The most efficient isolated fungus was identified to white-rot fungus Geotrichum candidum. Decolorization of OMW by Geotrichum candidum was investigated by using Hadamard's matrix for screening the important parameters and optimize them in order to control the biological decolorization. Agitation favours the conversion of COD removed into Geotrichum candidum biomass especially with high arthoconodia and few mycelium. Dilution of OMW and aeration enhanced the mycelium growth and rammification which that allowed polyphenols hydrolysis and then a decolorization. The initial pH of OMW is suitable for its decolorization by Geotrichum candidum growth. Ammonium sulfate concentrations tested with different OMW dilutions showed that the COD:N:S ratio of 100:5:2 is suitable for higher black colour removal. With optimized conditions Geotrichum candidum growth on OMW in laboratory scale bubble column, the OD removal reached 70% and all fractions of polyphenolic compounds of OMW were oxidized.     

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.     

Rhizosphere dynamics during phytoremediation of olive mill wastewater

The potential of phytoremediation as a treatment option for olive mill wastewater (OMW) was tested on five perennial tree species. Cupressus sempervirens and Quercus ilex proved tolerant to six-month OMW treatment followed by six-month water irrigation, whereas Salix sp. and Laurus nobilis and, later, Pinus mugo suffered from phytotoxic effects. Test plants were compared to controls after treatment and irrigation, by monitoring biochemical and microbiological variations in the rhizosphere soil. OMW-treated soils were exposed to 50-fold higher phenols concentrations, which, irrespective of whether the respective plants were OMW-resistant or susceptible, were reduced by more than 90% by the end of the irrigation cycle, owing to significantly increased laccase, peroxidase and β-glucosidase activities, recovery/acquisition of bacterial culturability and transitory development of specialized fungal communities sharing the presence of Geotrichum candidum. Of all results, the identification of Penicillium chrysogenum and Penicillium aurantiogriseum as dominant rhizosphere fungi was distinctive of OMW-tolerant species.     

Use of Geotrichum candidum for olive mill wastewater treatment in submerged and static culture

The production of lignin peroxidase (LiP), manganese peroxidase (MnP) and lipases by Geotrichum candidum were performed in order to control the decolourisation and biodegradation of olive mill wastewater (OMW). Optimisation of different factors showed that dilution, carbon and ammonium concentrations significantly affected decolourisation and activities of ligniolytic peroxidases (LiP and MnP) on OMW. Moreover, addition of olive oil and agitation improved the lipase production. Batch and continuous OMW treatments in settler or bubble column bioreactors showed high COD and colour removal efficiencies of 60% and 50%, respectively. Lipolytic activity was greater in the batch bubble column whereas, LiP and MnP productions were improved in the settler. The performance of the continuous processes decreased with the decrease of hydraulic retention time (HRT). It has been shown that decolourisation and biodegradation decreased with an average of 40% and 45%, respectively, by decreasing the HRT from 4 d to 1.7 d.     

Construction of a whole-cell catalyst displaying a fungal lipase for effective treatment of oily wastewaters

To combine the advantage of the oleaginous yeast Yarrowia lipolytica with the high activity of some fungal lipases for oily wastewater treatment, an effective lipase-displaying arming yeast was constructed using the flocculation functional domain of Saccharomyces cerevisiae as the protein anchor. To estimate the effect of the whole-cell oily wastewater treatment, the lipase-displaying arming yeast was added into an open activated sludge bioreactor. Within 72 h of whole-cell treatment, 96.9% of oil and 97.6% of chemical oxygen demand (COD) were removed, while only 87.1% of oil and 91.8% of COD were removed in control A (Y. lipolytica Polg was added), 45.1% of oil and 67.5% of COD were removed in control B (no cell was added) in 72 h. The lipase-displaying arming yeast exhibited remarkable oil removal and COD degradation effect compared with the control samples, exemplifying its application potential.     

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.     

Microbial Interactions within a Cheese Microbial Community

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.     

Aerobic biodegradation and detoxification of wastewaters from the olive oil industry

Growth and polyphenol biodegradation by three microorganisms namely Geotrichum sp., Aspergillus sp. and Candida tropicalis were studied on olive mill wastewater (OMW). These three microorganisms were selected for their tolerance to the polyphenols. The biodegradation process of OMW was investigated in batch regime by conducting experiments where the initial concentration of chemical oxygen demand (COD) was varied. Furthermore, some tests were performed to determine the most important nutrients necessary for aerobic degradation of OMW. Average COD removals were 55.0%, 52.5% and 62.8% in wastewaters fermented with Geotrichum sp., Aspergillus sp. and C. tropicalis, respectively. The maximum removal of polyphenols was 46.6% (Geotrichum sp.), 44.3% (Aspergillus sp.) and 51.7% (C. tropicalis). In addition, significant decolorization was evident.     

Optimization of the fermentation of olive mill waste-waters by Aspergillus niger

Summary The fermentation of olive mill waste-waters (OMW) by Aspergillus niger was studied. On the basis of factorial design experiments, suspended solids and concentration of OMW, nitrogen source, sulphate and size of inocula were all found to be significant by affecting mycelium growth and chemical oxygen demand (COD) removal. Neither the absence of yeast extract, magnesium, sodium, potassium nor of calcium limited the growth of A. niger. With media lacking additional nitrogen and sulphate, the growth was limited. The optimal inoculum obtained was between 10⁶ and 10⁷ spores/g COD. The highest biomass and the greatest COD removal were obtained with removed COD to N:SO _inf4 ^sup¨- ratios averaging 100 to 3:1.5. Offprint requests to: M. Hamdi     

Screening of antagonistic yeast strains for postharvest control of Penicillium expansum causing blue mold decay in table grape

Blue mold decay caused by Penicillium expansum is one of the most important postharvest diseases of grapes, leading to considerable economic losses. Regarding the increasing demand for pesticide-free foods, this study aimed to find potential yeast strains for biological control of blue mold on table grapes. A total of 50 yeast strains were screened for antagonistic activity against P. expansum using the dual culture method and six strains significantly inhibited the fungal growth. All six yeast strains (Coniochaeta euphorbiae, Auerobasidium mangrovei, Tranzscheliella sp., Geotrichum candidum, Basidioascus persicus, and Cryptococcus podzolicus) reduced the fungal growth (29.6–85.0%) and the decay degree of wounded grape berries inoculated with P. expansum while G. candidum was found to be the most efficient biocontrol agent. On the basis of antagonistic activity, the strains were further characterized by in vitro assays involving inhibition of conidial germination, production of volatile compounds, iron competition, production of hydrolytic enzymes, biofilm-forming capacity, and exhibited three or more putative mechanisms. To our knowledge, the yeasts are reported for the first time as potential biocontrol agents against the blue mold of grapes but more study is required to evaluate their efficiency related to field application.     

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.     

Immobilization of Leachable Toxic Soil Pollutants by Using Oxidative Enzymes

Screening of leachable toxic chemicals in a horseradish peroxidase-H₂O₂ immobilization system established that immobilization was promising for most phenolic pollutants but not for benzoic acid, 2,6-dinitrocresol, or dibutyl phthalate. The treatment did not mobilize inherently nonmobile pollutants such as anilines and benzo[a]pyrene. In a separate study, an extracellular laccase in the culture filtrate of Geotrichum candidum was selected from five fungal enzymes evaluated as a cost-effective substitute for horseradish peroxidase. This enzyme was used in demonstrating the immobilization and subsequent fate of ¹⁴C-labeled 4-methylphenol and 2,4-dichlorophenol in soil columns. When applied to Lakewood sand, 98.1% of 4-methylphenol was leached through with distilled water. Two days after immobilization treatment with the G. candidum culture filtrate, only 9.1% of the added 4-methylphenol was leached with the same volume of water. Of the more refractory test pollutant 2,4-dichlorophenol, 91.6% had leached at time zero and 48.5% had leached 1 day after the immobilization treatment. However, 2 weeks after immobilization, only 12.0% of the 2,4-dichlorophenol was leached compared with 61.7% from the control column that received no immobilization treatment. No remobilization of the bound pollutants was detected during 3- and 4-week incubation periods. Enzymatic immobilization of phenolic contaminants in soil appears to be a promising technique for the reduction of groundwater pollution by such substances.     

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.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

Assessment of olive-mill wastewater as a growth medium for lipase production by Candida cylindracea in bench-top reactor

Olive-mill wastewater (OMW) was investigated for its suitability to serve as a medium for lipase production by Candida cylindracea NRRL Y-17506. The OMW that best supported enzyme production was characterized by low COD and low total sugars content. In shake flask batch cultures, OMW supplementation with 2.4 g l⁻¹ NH₄Cl and 3 g l⁻¹ olive oil led to an enzyme activity of about 10 U ml⁻¹. The addition of glucose or malt extract and supplements containing organic N (e.g., peptone, yeast extract) either depressed or did not affect the enzyme production. Further experiments were then performed in a 3-l stirred tank reactor to assess the impact of medium pH and stirring speed on the yeast enzyme activity. The lipase activity was low (1.8 U ml⁻¹) when the pH was held constant at 6.5, significantly increased (18.7 U ml⁻¹) with uncontrolled pH and was maximum (20.4 U ml⁻¹) when the pH was let free to vary below 6.5. A stirring regime, that varied depending on the dissolved oxygen concentration in the medium, both prevented the occurrence of anoxic conditions during the exponential growth phase and enabled good lipase production (i.e., 21.6 U ml⁻¹) and mean volumetric productivity (i.e., 123.5 U l⁻¹ h⁻¹).     

Potential of pure and mixed cultures of Cladosporium cladosporioides and Geotrichum candidum for application in bioremediation and detergent industry

The effect of ethoxylated oleyl–cetyl alcohol (Henkel, “Merima”, Serbia) on the growth and metabolic activity of Cladosporium cladosporioides, Geotrichum candidum and their mixed culture was in the focus of this paper. The cultures were grown in Czapek-Dox liquid nutrient medium with the addition of 0.5% pollutant and without it. The physico-chemical and biochemical changes of pH, the total biomass dry weight, the quantity of free and total organic acids, proteolytic activity and the quality of carbohydrates were evaluated from 4th to 19th day of fungal growth. The pollutant caused an inhibitory effect on biomass dry weight of C. cladosporioides and G. candidum for 10.36% and 4.65% respectively, and stimulatory effect on biomass of mixed culture for 3.80%. The pollutant had influence on the decrease in pH value of the media in the phase of culture growth, and pH changes were correlated with the amount of excreted total organic acids. The highest quantity of free and total organic acids was noted in media with pollutant of mixed culture and C. cladosporioides, respectively. The alkaline protease activities of C. cladosporioides, G. candidum and mixed culture were enhanced by addition of pollutant for 56.88%, 55.84% and 30.94% respectively. The obtained results indicate the potential of both pure and mixed cultures in mycoremediation environment contaminated by alcohol ethoxylated and detergent industry.     

尾叶桉林下5种植物的耐阴性、生物量及其个体消长

为了解尾叶桉林下不同植物种类的生理生态特性和生长适应性,在鹤山站尾叶桉林中选取分布较多且药用价值和经济价值较高的5种林下植物为研究对象,测定各植物的叶片特征、叶绿素含量和光合-光响应曲线,同时结合2006年和2011年尾叶桉林的样方调查数据分析了5种林下植物生物量的变化和个体数量的消长。结果表明:(1)野牡丹叶片的宽度、厚度、单片叶面积等指标最大,梅叶冬青最小,桃金娘、栀子和山苍子处于两者之间;桃金娘、野牡丹和山苍子的光合速率随光照的增强迅速增大,其光饱和点较高;梅叶冬青和栀子的光合速率也随光照的增强而增大,但其斜率比野牡丹和山苍子大,并很快就达到光饱和点。综合聚类分析植物的耐阴程度,5种植物可分为2类:第1类为耐阴性较强的梅叶冬青、栀子,第2类为耐阴性较弱的野牡丹、山苍子和桃金娘。(2)随着尾叶桉林的生长,5种林下植物的生物量都有不同程度的增加,其中增幅较大的是野牡丹和桃金娘分别增加74倍和18倍,增幅最小的是栀子仅1.3倍。野牡丹、桃金娘和山苍子这3个种生物量的增加主要是由个体高度和地径的增长引起,而栀子和梅叶冬青生物量的增加是其个体数量、株高和地径做出的贡献。(3)在尾叶桉林生长过程中,耐阴种类梅叶冬青随着个体密度、株高和地径的增长,其在林内发展成为主要优势灌木种类;栀子的密度虽然有一定的增大,但其株高和地径增加量小,生物量也小,发展为次要灌木种类。野牡丹、桃金娘和山苍子等3种的个体密度都在下降,虽然其地径、株高和生物量也有一定的增长,但其较强的阳生性不能适应林内的荫蔽环境,只适于林窗或林缘光照较强的地方生长。因此,在桉树林经营管理过程中应适当保护和种植梅叶冬青和栀子;在短轮伐期经营模式下,林窗和林缘也可适当保留野牡丹、桃金娘和山苍子。     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.