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.     

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.     

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.     

Integrated biological process for olive mill wastewater treatment

The biological process for OMW treatment is based on an aerobic detoxification step followed by methanization step and aerobic post-treatment.The first aerobic detoxification step of OMW supplemented with sulfate and ammonium was carried out by the growth of Aspergillus niger in a bubble column. This step decreased OMW toxicity and increased its biodegradability because of phenolic compounds degradation. Growth of A. niger resulted in 58% COD removal, with production of biomass containing 30% proteins (w/w). Filtration of OMW was enhanced by this fermentation because the suspended solids were trapped in the mycelium. The filtrate liquid was then methanized using an anaerobic filter packed with flocoor. This reactor showed a short start up and a good stability. COD removal was around 60% and the methane yield (1 CH₄/g COD removed) was close to the theoretical yield.The anaerobic filter effluent was treated in an activated sludge fluidized reactor containing olive husk as a packing material. Husks were maintained in fluidization state by the aeration. This step induces COD removal at 45% and sludge (up to 2 g/dm³).The entire process allowed a global COD reduction up to 90%; however, the black colour due to polyphenolic compounds with high molecular weight persisted.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

Effect of agitation and aeration on the reduction of pollutant load of olive mill wastewater by the white-rot fungus Panus tigrinus

The white-rot fungus Panus tigrinus CBS 577.79 was cultivated both in mechanical (stirred tank, STR) and pneumatically (bubble column, BCB) agitated bioreactors and investigated for its ability to reduce the polluting load of olive-mill wastewater (OMW). Both aeration and agitation strongly influenced treatment efficiency. Best pollutants biodegradation performances were achieved in the bubble column bioreactor. Using this bioreactor, COD reduction, dephenolization and decoloration were 60.9, 97.2 and 75%, respectively. In contrast, lower depollution efficiency was generally observed in STR due to the possible occurrence of shear stress.     

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     

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.     

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.     

Decolorization of dye and molasses by continuous and semi-continuous jar-fermentor cultures ofGeotrichum candidum Dec 1

Two culture modes, continuous and semi-continuous, of the decolorization fungus,Geotrichum candidum Dec 1, were compared to obtain a high treatment efficiency of molasses decolorization and a large productivity of peroxidase (DyP) to simultaneously decolorize dyes and molasses. The continuous culture ofG. candidum Dec 1 using a 5-l jar-fermentor showed high DyP activity at a low dilution ratio of 0.005h⁻¹, and decolorization ratio of molasses of 80% was obtained concomitantly. Therefore, a semi-continuous culture was performed by repeated refill and draw. In this mode, approximately 1.5 liters of the culture broth was replaced per cycle when the decolorization ratio of molasses was near 80%. The molasses medium (1.0 liter per day) was treated and the peroxidase productivity in the drawn culture broth was 26.6 U/day, whereas the peroxidase productivity was 17.9 U/day in the continuous culture with a dilution rate of 0.005 h⁻¹. The semi-continuous treatment system was an efficient decolorization method for the strain,G. candidum Dec 1.     

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.     

Comparative effect of different aerobic pretreatments on the kinetics and macroenergetic parameters of anaerobic digestion of olive mill wastewater in continuous mode

A comparative kinetic study was carried out on the anaerobic digestion of olive mill wastewater (OMW) and OMW that was previously fermented with Geotrichum candidum, Azotobacter chroococcum and Aspergillus terreus. The reactors used were continuously fed and contained sepiolite as support for the mediating bacteria. A kinetic model for multicomponent substrate removal by anaerobic digestion has been used. The model is based on the linear removal concept which is a special case of the broader Monod equation. The second-order kinetic constant, k _{2( s )}, was found to be influenced by the pretreatment carried out, and was 4.2, 4.0 and 2.5 times higher for Aspergillus, Azotobacter and Geotrichum-pretreated OMWs than that obtained in the anaerobic digestion of untreated OMW. This was significant at 95% confidence level. This behaviour is believed to be due to the lower levels of phenolic compounds and biotoxicity present in the pretreated OMWs. In fact, the kinetic constant increased when the phenolic compound content and biotoxicity of the pretreated OMWs decreased. In addition, the macroenergetic parameters of the anaerobic digestion of OMW, i.e. the specific rate of substrate uptake for cell maintenance, m, and the yield coefficient for the biomass, Y, decreased by a factor of 2.4, 3.6 and 5.1 and increased by a factor of 1.9, 2.2 and 2.4 respectively, for the OMWs previously treated with Geotrichum candidum, Azotobacter chroococcum and Aspergillus terreus in relation to the observed values for the untreated OMW.     

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.     

Carbon and nitrogen yields during batch cultures of Geotrichum candidum and Penicillium camembertii

Batch cultures of Geotrichum candidum and Penicillium camembertii were carried out on peptones as carbon and nitrogen source and in the presence of lactate as a second carbon source. Unless growth ceased, carbon and nitrogen yields remained constants, except yields involving lactate consumption by G. candidum, since this fungus preferentially metabolized peptones as a carbon source. For both fungi, nearly 40% of the available carbon was metabolized for cellular biosynthesis and the remainder (about 60%) as carbon dioxide, for the energy supply of both biosynthesis and viable cell maintenance. Moreover, in relation to their carbon content, amino acids contain excess nitrogen, which was released as ammonium. From all these, the yields of ammonium nitrogen on cellular nitrogen were in all cases higher than 1, and were especially high when the medium contained only peptones as a carbon source, 4.4 and 5.7 for G. candidum and P. camembertii respectively. Indeed, in this case, the excess nitrogen was especially pronounced.     

Improvement of fermentative decolorization of olive mill wastewater by Lactobacillus paracasei by cheese whey's addition

The effect of the cheese whey's (CW) addition on the fermentative decolorization of olive mill wastewater (OMW) by Lactobacillus paracasei, with and without pH adjustment by lime, was investigated. Mixtures OMW/CW at different proportions were fermented. The highest colour removal (47%) and total phenolic reduction (22.7%) of OMW were obtained after cofermentation of OMW/CW at proportions of 10/90, respectively. The decrease of pH after cofermentation of the two wastewaters, induced the precipitation of whey proteins with phenolic compounds and, so, improves decolorization. These removal yields reached 64% and 34%, respectively after precipitation by adjustment of pH at 7 with lime at the end of cofermentation. These improvements were correlated to a clarification of wastewaters by precipitation of whey proteins with phenolic compounds. An enhanced decolorization (up to 93%) and a total phenolic reduction (50%) of the mixture were obtained when cofermentation sequentially pH corrected by lime addition was investigated.     

Involvement of lignin peroxidase in the decolourization of black olive mill wastewaters by Geotrichum candidum

AIM: Decolourization of black olive mill wastewaters (OMW) by depolymerization of phenolic compounds by Geotrichum candidum. METHODS AND RESULTS: Our results show that G. candidum is able to grow on black OMW supplemented with carbon source and nitrogen. The Geotrichum growth decreased the pH and induced a 49% of colour removal when the black OMW was supplemented with glycerol and diammonium tartrate (20 mm ammonium). An improvement of 10% of colour removal was observed when the culture was supplemented with veratryl alcohol. The decolourization was inhibited with glutamate as nitrogen source. CONCLUSION: Our results suggest the potential use of G. candidum in black OMW decolourization and support the concept that lignin peroxidase (LiP) of G. candidum is involved in the depolymerization of phenolic compounds. Significance and Impact of the Study: This is the first report of LiP production by G. candidum on OMW.     

Characteristics of a newly isolated fungus,Geotrichum candidum Dec 1, which decolorizes various dyes

A fungus, Geotrichum candidum Dec 1, newly isolated from soil as a dye-decolorizing microorganism, decolorized 18 kinds of reactive, acidic and dispersive dyes and 3 model compounds on a solid medium, showing a broad spectrum of decolorization. Except for dispersive dyes, all the dyes used on the solid medium were also decolorized even in a liquid medium, although the decolorizing rates varied depending on the dye structure. By repeated addition of one dye, Reactive blue 5, about 12 g/l of the dye was degraded without significant decline of activity, showing the resistant property of Dec 1 to a high concentration of the dye. An energy source and oxygen were essential for the expression of decolorizing activity; the optimal temperature was 30°C. A crude extracellular enzyme solution, in which the decolorizing activity was more than 100 times that of the Dec 1 culture broth, showed peroxidase activity, indicating that some peroxidases are responsible for dye-decolorization.     

Growth of Geotrichum candidum and Penicillium camemberti Cultivated on Liquid Media Correlated with Ammonia and Methanethiol Emission

The growth of Geotrichum candidum and Penicillium camemberti plays an important role in the ripening of Camembert‐type cheeses, but the monitoring of the corresponding kinetics for fungal cocultures on solid media appears difficult. Continuous and non‐intrusive methods to characterize the growth of both species (like the monitoring of the emissions of ammonia and volatile sulphur compounds) may be highly relevant, under the condition that such emissions could be correlated with growth. This would be easier to investigate in submerged culture, since total biomass concentration is known to vary in proportion to broth turbidity. For this reason, growth kinetics, ammonia and flavour gas emission of both Geotrichum candidum and Penicillium camemberti grown separately in submerged cultures under the conditions of low aeration rate and uncontrolled pH were continuously recorded.In the basal medium (peptone+lactate supplemented with both glutamic acid and methionine [1 g/l]each), no significant gas emission was observed during the growth of both fungi. Ammonia and sulphur gas emissions by G. candidum were a little stimulated by supplementing the basal medium with trace elements, and, at a larger extent, by the addition of inorganic phosphate: Such a gaseous emission took place at the end of the growth phase of G. candidum. Irrespective of the basal medium supplementation, no significant emission ofammonia and sulphur gas was observed during the growth of P. camemberti. For the media and strains used, ammonia and volatile sulphur compounds emissions unequivocally showed the growth of Geotrichum candidum.     

Repeated batch process for biological treatment of black liquor using brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis</Emphasis> sp. IMER2

A repeated batch operation is developed for the treatment of alkaline pulp black liquor, through a process of biological acidification precipitation of lignin using brown rot fungus Fomitopsis sp. IMER2. The results showed that COD and color removal of black liquor was dependent on the biomass concentration, pH decrease and initial COD. Based on these results, the repeated batch process was successfully carried out 12 times over 36 days in an air bubble column bioreactor. The average reduction of COD and color was approximately 40% and 70%, respectively.     

Sporulation and the Macromolecular Composition of the Mycelium and Arthrospores of Geotrichum candidum

In submerged culture the mycelium of Geotrichum candidum breaks into fragments (arthrospores) which are either cylindrical or ellipsoidal in shape; the proportion of each spore type depends upon the glucose concentration in the medium. Above 0.2% glucose the ellipsoidal type prevails, whereas the cylindrical type is more abundant at lower glucose concentrations. The cylindrical spores and the mycelium have a very similar macromolecular composition, but the ellipsoidal spores have less RNA and protein and more carbohydrate than the mycelium.