bioremediation of alpechin

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Intemational Biodeterioration & Biodegradation (1995) 249-268 Copyright 0 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved

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Bioremediation of Alpechin

A. Ramos-Cormenzana, M. Monteoliva-Sanchez & M. J. Lopez

Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain

ABSTRACT

Olive oil extraction produces large amounts of residues. These olive-mill wastes are known as alpechin. Alpechin-polluted waste waters are resistant to degradation. They are regarded as a severe environmental problem because of their high organic content, largely simple phenolic compounds that are both antimicrobial and phytotoxic.

During the past years, most of the investigations related to alpechin treatment focused on the microbial degradation of the alpechin polyphenols.

However, the actual perspectives are based on both the bioremediation of alpechin and on the modification of the technology used in olive-oil extraction. In this article we will describe the most relevant biotreatment systems that can be used for the recycling of alpechin wastes. We will also discuss

for each system their prospects for future uses. The various systems we will discuss include the following: (I) Bioremediation for use as fertiliser or soil conditioner. (2) The utilisation as a medium for grown edible mushrooms. (3) The application as a growth medium for algae in open basins. (4) Biopolymeric substances production from alpechin, focusing on poly- sacharide and biodegradable plastics production. (5) The use as a bioenergetic source (or for biogas production). (6) The employment of alpechin as a source of biopharmaceuticals.

INTRODUCTION

Olive oil extraction produces a large volume of solid and liquid residues, the olive-mill waste waters are known as alpechin (Fig. 1). The world production is widely distributed, but the two most important olive oil producers are Spain and Italy (Fig. 2).

249

250 A. Ramos-Cormenzana, M. Monteoliva-Sanchez, M. J. Lopez

WATER 1.250

WATER 1.375Kg

Fig. 1. Olive by-products.

Olive oil waste effluents are major pollutants and constitute a great problem in olive tree cultivation areas. In several European countries including Italy and Spain, it is illegal to dispose of the wastes directly into waterways, and they have to be stored in closed or open ponds.

Closed ponds generate large amounts of sludge, which has to be disposed. These types of closed ponds for alpechin storage are sources of contamination for surface and ground waters.

In the case of open ponds, the water is evaporated by sunlight all year long, and a sludge is created, which must be disposed of. This method needs large areas of arable land and is generally insufficient in all respects.

Open and closed ponds generate many volatile malodorous compounds. Both systems are very costly due to high labour and maintenance expen-

SPAIN 41%

PORTUGAL 2%

Fig. 2. European Community olive oil production.

Bioremediation of alpechin 251

ses. Moreover, in these systems, the water and the valuable substances contained within the wastes which represent potential financial incomes are lost.

During past years, efforts were made to seek a viable solution to solve the problem and the overall objectives of most research programs focused on strategies that would help increase the commercial potential of alpechin. The first investigations deal with the microbial degradation of the alpechin polyphenols. In order to prevent industrial pollution, new highly sophisticated treatment plants are built in polluted geographical areas. The schemes presented on Figs 3 and 4 illustrate strategies that are proposed to reduce alpechin generated pollution. Some people believe that the two-phase olive oil production system shown on Fig. 4 will solve the pollution problem. However, this system generates a new waste product, called Alpeorujo in Spain which will have to be treated. In addition, the two-phase process is very costly.

The problem concerning the alpechin is derived from its composition. Many references can be found on this subject-Moreno et al. (1990), Ramos-Cormenzana (1986), Fiestas Ros de Ursinos (1958) and others. The fundamental composition is essentially water, organic substances (organic acids, phenols - which are considered responsible for the toxic properties - miscellaneous organic compounds) and minerals. However, the alpechin composition is highly variable, when considering each individual component, mainly because of the fact that the alpechin

u ELIMINATE ) 4 w

Fig. 3. Solutions proposed for the alpechin environmental problem.


OLIVE OIL SYSTFM PRODUCTION QLIVE OIL TWO PHASES SYSTFM PRODU- -k

d OLIVE

io,,, &

OIL

Fig. 4. Olive oil extraction technology.

is a natural product, processed from a raw material and subject to varying conditions which are difficult to control. In this sense the forthcoming new technologies should take this into account. The olive oil waste water is resistant to degradation and represents a severe environmental problem because of its high organic content, largely simple phenolic compounds (Gonzalez et al., 1990) that are both antimicrobial and phytotoxic. Therefore, new alternatives are actually needed for their disposition.

However, not all the effects of alpechin are adverse and there are some important beneficial effects. For example, olive oil waste waters also contain valuable substances which could be recovered. Some of these can stimulate the soil nitrogen-fixing bacteria population or the production of extracellular slime which contributes to the stability of soil aggregates and hence the tilth of the soil and its water-retention capacity. Other components of these wastes can suppress soil-borne fungal foot pathogens, such as Pythium, Phytophthora spp.

Moreover, these waste products could be used as a substrate in biotechnological processes, for the production of other valuable metabo- lites. A solution for the treatment and disposal of alpechin therefore should not only consider the environmental aspects, but also the economic potential of valuable substances it contains, including residual oil. In this case, income from valuable products should make up for the cost asso- ciated with treatment and disposal.

In Mediterranean countries, constraints imposed by the strict environmental protection regulations which will be enforced in the EEC member states will also have be considered for proper utilization of these effluents.


The new perspectives for use of oil mill waters and olive residues have been reviewed by different authors of various countries such as Italy (Fedeli, 1986) Greece (Israelidis, 1990), Tunisia (Friaa et al., 1986) and Spain (Ramos-Cormenzana, 1987).

In this article, some of the biotechnical solutions (processes) proposed today for partial treatment and disposition will be discussed briefly. Some of these processes are still at an experimental level and have never been used on a large scale.

Although a fairly large number of potential uses of alpechin have been proposed, in order to avoid going into details for all the different aspects, we will only take into account the following uses (Fig. 5):

(1) Its use as a fertiliser. (2) Its use as food and in the food industry. (3) Its utilisation as a growth medium for algae. (4) Biopolymeric substance production from alpechin focusing on

polysaccharide and biodegradable plastics. (5) Its use for bioenergy production. (6) The employment of alpechin as a source of biopharmaceuticals.

In one sense it can be said that the problem concerning alpechin decontamination has been solved, but serious economical problems still remain. Fedeli (1986) made an excellent study dealing with the economic aspect of alpechin decontamination and utilization. He considered five parameters: processing costs, investments, waste cleaning costs, residual ecological costs, and products value. All these factors need to be considered to evaluate the feasibility of the various processes that are proposed and to classify them following an order of priority.

r\ :- . BIOPHARMAGEUTICALS

FERTILISER

bE :ENE)

FOOD AND THE BIOENERGETICS FOOD INDUSTRY

Fig. 5. Bioremediation of alpechin.


The equation represents these factors is the following:

CE+CI+CD-RP=Q,

where:

CE = ecological costs; CI = industrial costs; CD = waste cleaning costs; RP = product value; Q = profitability evaluation factor.

Any kind of solution should take into account the above considerations.

BIOREMEDIATION FOR USE AS A FERTILISER

The aqueous wastes from olive mill pressings represent a valuable fertilis- ing material and a good source of organic compounds, which hitherto have not been fully exploited, because the wastes are produced in very large amounts during the brief rainy season, they undergo rapid biodegradation and are toxic to plant and microbial life when applied to agricultural lands in large quantities. The most prominent adverse effect is its phytotoxicity that is largely attributable to the phenolic compounds of alpechin. Indeed, when applied to soil, alpechin acts like a broad-spectrum herbicide, where its half-life depends on the amounts applied to soils and the frequency of application in a given period on the soil aeration. In Spain, as we have previously pointed out, it is illegal to dispose of the wastes directly into waterways, and there are serious concerns that use of unremediated alpechin as a fertiliser will cause pollution of water courses.

Use as a soil conditioner

As alpechin contains large amounts of minerals (potassium) and organic matter (Fig. 6), its use as a fertiliser was tested after prior neutralisation with lime and it gave satisfactory results in experimental corn and wheat farming. The experiments carried out indicated that it is advisable to use approximately 11 of alpechin/m* (Albi Romero & Fiestas Ros de Ursinos, 1960).

Direct application of olive oil liquid waste onto soil was found to stimulate the respiratory activity of the soil along with the nitrogen-fixing capacity. Chatjipavlidis et al., (1986) reported that repeat applications result in an enhancement of the microbiota which can degrade the phytotoxic components of the liquid wastes. These authors claimed that their treatment could stabilize the soil aggregates.

The effect of alpechin on the bacterial population of soil has been investigated (Paredes et al., 1986; Moreno et al., 1987), and also the


ORGANIC MATTER 15% INORGANICS (1.8%):

POTASSIUM 0.85% SODIUM 0.13% NITROGEN 0.2%

COMPOSTING DIRECT APPLICATION

Fig. 6. Alpechin use as a fertiliser.

microbial feature of soil after pollution with oil mill waste waters (Paredes et al., 1987) - a decrease in the count of aerobic sporulated bacteria was noted.

No economic evaluation of this approach has yet been made since it is still under investigation and has not reached the pilot plant scale.

Although this approach is ecologically interesting to solve the alpechin problem, unfortunately it does not allow the recovery of the valuable substances contained in the liquid wastes.

In Spain, as ecological agriculture is becoming very popular, in this sense alpechin conveniently prepared could be used as a substitute for chemicals. The Spanish government is preparing new environmental regulations that will probably include the possibility of using fertilisers obtained from alpechin.

Our group is presently working on a project dealing with the bioremediation of olive-mill wastes for use as a fertiliser and whose objective aims at understanding of the basic microbiological and biochemical processes required to improve recycling/detoxication composting processes (Russell, 1994).

Physical and chemical processes to purify or detoxify alpechin have met with limited practical success and were not shown to be cost-effective. Microbial conversion appears to represent the only viable alternative. Indeed, microbial isolates not only able to resist the high osmolarity, elevated temperature and toxic nature of alpechin, but which are also able to break down the major toxic alpechin components, namely phenolics and fatty acids, have been isolated. Moreover, the variety of organic components of alpechin favors the development of microbial consortia endowed with a broad array of metabolic capabilities that are useful in the


bioremediation process of alpechin. The inclusion of nitrogen-fixing species in the consortia has the added advantage that the final product has a better mineral balance and thus a greater fertilisation potential.

These investigations have shown that the compost obtained by cornposting olive-mill wastes with other organic wastes can be used as fertiliser and soil conditioner. All the alpechins are useful for compost preparation, although their N and P contents were very low, as was the level of micronutrients. Plant wastes (carriers) can be quite suitable for preparing organic fertilisers, because of their high organic content. Also, sufficient amounts of N were found in some of the carriers (sugar cane sludge, spent mushroom compost, cotton waste and grape mart). Humic- like substances are generated during the alpechin cornposting, as a result of the increasing humilication of the organic matter. The humic and fulvic acids (HA and FA) concentration varied according to the method used. The highest HA content was found in compost obtained by mixing 654% of cotton waste with 34.6% of fresh poultry manure (weight), with fresh alpechin at a rate of 1930 l/ton being added to the solid mixture during the 18 days of cornposting. The lowest levels of HA were found in compost obtained by mixing sewage sludge with fresh alpechin. The HA content generally decreased in the mature composts as compared with that found in the thermophilic phase compost. The HA/FA ratio remained nearly constant during cornposting [Cegarra, 1994, in Russells (1994) report].

Several reports suggest that nitrogen-fixing organisms increased after soil treatment with olive-mill wastes (Garcia-Barrionuevo et al., 1992, 1993). Therefore, it is likely that alpechin could be used as a substrate for nitrogen fixation (Perez & Gallardo-Lara 1987; Balis & Chatjipavlidis, 1994), and further study should be of great interest.

However, although use of alpechin as a fertiliser appears a viable alternative, other bioremediation methods should be used because this process alone would not be sufficient to cope with the enormous quantity of alpechin produced.

USE AS FOOD AND IN BIOMASS AND SINGLE CELL PROTEIN FOOD INDUSTRY (Fig. 7)

It is known that most research is either done in the industrialised countries, or is influenced by their needs; this is the case of the alpechin substrate that could be used to produce yeast for use as feed or fodder. The production of microbial biomass for use as single cell protein will be economically viable if the costs are low for large-scale production. Using Torulopsis utilis, Fiestas Ros de Ursinos (1958) has shown that the yeast


EDIBLE MUSHROOMS

SainzJtmenez 8 Gomez-Alarc6n. 1966

Fig. 7. Food and food industry uses for alpechin and derivatives.

Codounlsel a., 1963

could grow sufficiently well in chemostat on alpechin to use the process for industrial Torulopsis yeast production.

Mention should also be made of the observation that many kinds of edible mushrooms are growing in composts fed with alpechin as substrate (personal communications, Balis & Tomati).

In fact, the effects of alpechin on fungi has been investigated by Saiz- Jimenez & Gomez-Alarcon (1986). They determine the effect of alpechin on 19 imperfect fungi. With the exception of Chaetomium &turn and Inotus hispidus, which were completely inhibited, the other species studied were able to grow on malt extract, although with a small delay with respect to the control plates. Other studies have also been made on using olive milling waste-waters as a medium for growth of Pleurotus spp. (Sanjust et al., 1991).

The isolation of thermophilic fungi (Thermoascus, Mucor, Tuluromyces, Torula, and Humicola) from co-composting material of exhausted olive husk and oil mill waters has been reported by Demou & Balis (1994). Although it needs to be confirmed, the same authors have more recently isolated Coprinus species from similar material (Russell, 1994).

Recovery of feed additives

Natural pigments (anthocyanins) contained in the alpechin have been reported by several authors. Codounis et al., (1983) developed a process for the extraction and purification of anthocyanins from alpechin. According to this process the eluent from olive extraction plants is first passed through an ultrafiltration unit which separates the proteins and high molecular weight carbohydrates from simple sugars, anthocyanins,

258 A. Ramos-Cormenzana. M. Monteoliva-Sanchez, M. J. Lopez

phenolic substances, vitamins, aminoacids and minerals. The concentrate, which is retained on the filter, is rich in proteins and carbohydrates. It can be used, after fermentation, as a liquid feed supplement or it may be dried and fed to animals.

The extraction and purification of anthocyanins contained in the permeate can be achieved by chromatographic separation on ion exchange resins. These anthocyanins can be used as natural food colouring agents,

The antioxidant activity of alpechin phenols could also be exploited for various applications in the food industry.

THE UTILISATION AS GROWTH MEDIUM FOR ALGAE

In a study on the microbial characterisation of effluent waters from olive oil mill (Ramos-Cormenzana, 1986), it was found that a large spectrum of microorganisms could grow on agar-solidified medium made with the effluent waters as culture medium. It is thus suggested that because of the high temperatures prevailing in southern Spain, lagoons containing effluent waters from olive oil could be used to grow algae. Lagoon temperatures fluctuate depending on liquid depth and ambient temperature. However, by controlling the waste water flow in these lagoons it is expected that the temperature would be controlled. These lagoons could be inoculated with species such as Dunaliella or Spirulina. Ramos-Cormen- zana (1989) has already shown that Dunaliella can grow on alpechin medium. The salinity prevents the invasion and development of other microorganisms. Thus, the possibility is that under seminatural conditions, or in ponds, Dunaliella could grow in lagoons to produce biomass and b-carotene.

It has been suggested that biomass yields for Scenedesmus cells grown in the Mediterranean area should be of the order of 30-32 g/m2 day (Tamiya, 1975). Efforts should be made to study the ability of these algae to grow on alpechin medium in seminatural conditions.

BIOPOLYMERIC SUBSTANCE PRODUCTION FROM ALPECHIN, FOCUSING ON POLYSACCHARIDES AND BIODEGRADABLE

PLASTICS PRODUCTION

Ragazzi & Veronesse (1967) were the first to describe a brownish-black coloured polymer present in alpechin. Since then, the polymeric substances have been character&d in fresh vegetation water and sludge evaporation ponds (Saiz-Jimenez et al., 1986; Sanchez-Lama, personal communication).


PHB (poly-hydroxy-b-butyric acid) and exopolysaccharides have been detected in certain microbial cultures. Studies are in progress in our laboratory to further characterize these biopolymers (Fig. 8). Two of these polysaccharides are really interesting: pullulan and xanthan.

Pullulan is an extracellular polysaccharide produced by the fungus Aureobasidium pullulans. It is a marketable product of high value and it is used in both food and pharmaceutical industries. Olive oil liquid wastes were shown to provide good quality organic substrate for pullulan production fermentation. Preliminary experiments which were done at the Institute of Agricultural Products Technology in Athens, showed that pullulan was produced at yield of 8 g/l, with a ratio of bioconversion of total sugars of O-5 g of pullulan/g total sugars, when the liquid wastes had been diluted to an extent of three parts liquid wastes for one part water.

Investigations in our laboratory on pullulan production has provided a promising result. A relatively high conversion rate of alpechin to polysaccharide xanthan and pullulan were obtained. The optimal alpechin concentration for growth and pullulan production by Aureobasidium pullulans was 70% (vol/vol), providing pullulan production yield above 3 g/L (Quevedo-Sarmiento et al., 1991). A detailed study on xanthan formation in cultures of Xunthomonus campestris, S4LLIA and Xanthomonas campestris, 646A showed the ability of these microorganisms to grow and produce xanthan in a medium containing 3&50% vol/vol alpechin (Lopez-Lopez, 1993).

The use of alpechin as a substrate to exopolysaccharides production by

0 PULLULAN j

HALOPHILE

A I

u!J

PHB

Fig. 8. Biopolymers obtained from alpechin.


moderately halophilic microorganisms has also been assayed in our laboratory. The rationale for these experiments was based on the facts that several halophilic bacterial strain were shown to degrade a variety of phenolic compounds (Yanase et al., 1992) and that several moderately halophilic bacteria are shown to be good producers of exopolysaccharides. Promising results were obtained working with VolcunieZlu eurihdina, a moderately halophilic bacterium described by us (Quesada et al., 1990), that produces an extracellular polysaccharide in very large amounts, whose chemical and physical properties could be of interest in a number of industrial applications. The exopolysaccharide production was investigated first using MY medium (Moraine & Rogovin, 1966), and afterwards using MY medium modified by the incorporation of different amounts of salts (up to 10%) and alpechin (up to 40%). The EPS was isolated and purified, the EPS synthesis increased with increasing salt concentration up to 7.5% (wt/vol); above this concentration the productivity dropped drastically. In the absence of salt, alpechin was also found to increase EPS production.

PHB and PHA

In a recent study on the effect of olive oil mill waste-waters on Azoto- batter, it was observed that it stimulates PHB production by Azotobacter chroococcum cells (Garcia-Barrionuevo, 199 1). More recently, Martinez- Toledo et al. (1994) studied the PHB formation from alpechin using Azotobacter chroococcum H23 strain. Therefore, alpechin could most probably serve as an inexpensive substrate for PHB, thereby increasing the likelihood of producing bioplastics at competitive prices.

USE AS A BIOENERGETIC SOURCE (OR FOR BIOGAS PRODUCTION)

Biogas

Fermentation to produce biogas has been common practice in urban waste water treatment plants for many years now. Compared to other existing technologies, anaerobic treatment counts among those that can reduce the amount of pollutants at a reasonable cost.

The final olive-mill waste waters, when tried as fertiliser, had kept its phytotoxicity practically unchanged and, as described by Ioniotakis et al. (1990), such highly toxic waste water cannot be purified with biological methods. As with other types of anaerobic treatment, methane and carbon


dioxide are two of the degradation by-products. Alpechin is a good potential energy source for anaerobic fermentation because of its high organic content. Methane is a potential of biogas (Rozzi & DiPinto, 1986; Carrieri et al., 1986); however, the production costs for biogases are often rather high.

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats, which are designated metha- nogenic bacteria. Several studies have already been carried out to evaluate the possibility of using alpechin for methane production (Curi et al., 1988; Martin et al., 1991).

The advantage of biogases is their further potential uses as energy sources. For this reason biogas production processes were retained among the research priorities at the Instituto de la Grasa y sus Derivados, Sevilla.

Results obtained in Greece (Dal&, 1989; Georgakakis, 1989) have shown that biogas was produced from olive mill waste wasters, under anaerobic fermentation, giving yields of 1.5 m3 per m3 of available volume in the bior- eactors. The fermentation resulted in a sharp drop of the initial BODs where most of the major groups of organic compounds were affected.

However, although substantial investments were made to try to opti- mise this process, so far the results have been less encouraging than was expected. One of the major problems is most probably related to the influence of the alpechins, which are toxic or inhibitory to microorganisms both under aerobic and anaerobic conditions. The major effect of phenolic acids from alpechin on microbial consortia involved in the methanogenesis of olive mill waters have been investigated (Sorlini et al., 1986).

Pretreatments of alpechin to reduce the phenolic compounds through aerobic biodegradation processes using Aspergillus terreus (Martinez- Nieto et al., 1992, 1993), or Azotobacter chroococcum (Borja et al., 1993), were shown to substantially improve methane production, when compared to methane production during anaerobic digestion of untreated olive mill waste waters.

Production of ethanol

Production of other bioenergetic compounds from alpechin (Fig. 9) has also been evaluated. Butanol, butanediol (Wachner, Mendez & Giolietti, 1988) and ethanol (Bambalov et al., 1989a, b) counts among the compounds tested.

Ethanol can be produced using olive oil waste effluents as a substrate. The microbial strains involved in these processes have been isolated from such effluents and they were tentatively identified as Candida wickerhamii, C. molischiana and Saccharomyces cereviae (Bambalov et al., 1989a).


IOENERGETICS

Fig. 9. Bioenergetics products obtained from alpechin.

The product yields (g of ethanol per g of sugar) reached 87% of the theoretical yield, but the final alcohol concentration was low, about 1.3% at the very best (Bambalov et al., 19893).

The recovery costs for such a low concentration of ethanol from the fermentation broth are likely to be rather high if traditional extraction methods are used. However, the process may be less expensive if other methods such as membrane separation could be used for extractions.

THE EMPLOYMENT OF ALPECHiN AS SOURCE OF BIOPHARMACEUTICALS (BIOLOGICAL AND PHARMACOLOGICAL ACTIVITIES) (Fig. 10)

It is known that a number of compounds found in alpechin are inhibitory for microorganisms (Gonzalez et al., 1990). Although the use of such compounds as antimicrobials in pharmacological applications has not yet been exploited, it is certainly to be considered.

On the other hand, various alpechin components could be extracted for specific applications. Polyphenols represent such a class of compounds that could have interesting pharmaceutical applications. We are aware that many research projects intended to exploit these compounds are going on. More complex separation strategies to recover alpechin components separately have been suggested. Unfortunately information pertain- ing to these projects is kept secret by the industries supporting those projects.

However, I will describe some of these useful applications, based on published works, personal observations or personal communications.


ANTIBIOTICS

BIOLOGICAL RESPONSE MODIFIERS

BIOPHARMACOLOGICAL ACTIVITY1

NEWS PHARMACOBIOLOGICAL MOLECULES

Fig. 10. Biopharmaceutical uses of alpechin

The antibiotic production is one of the more feasible applications, based on the known antimicrobial activity of alpechin. More properly tetra- cycline could be among alpechin components.

The polyphenols could be obtained from the alpechin and they could be used in similar applications than other polyphenols compounds that have been tested for their pharmacological activity. Particularly they are good antifungal compounds. It has also been suggested that these compounds could be used for the control of a number of fruit and vegetable diseases (Lattanzio et al., 1994).

Recently, vegetal polyphenols were considered as potential antiviral compounds (Buciumeanu et al., 1994). Based on its chemical composition I dare to comment that in a similar way, alpechins polyphenols might also be considered for this application.

In the last XVIIth International Meeting of the polyphenols group, Takashi et al. (1994) pointed out that ellagitannin oligomers have antitumoral activities. Based on the relationship between the whole structures of these oligomers and some that could be found in alpechin, we suggested that it would be worthwhile to study the possible application of oligomers from alpechin as biological response modifiers. Similarly investigations on polysaccharides obtained by alpechin biotransformation would be interesting to initiate. The organic acids should also be studied as biological response modifiers.

Other possibilities should not be excluded and enzyme inhibition assays are valuable screening tools for the detection of the bioactive action of alpechin extraction compounds. Their biological activities are some times very difficult to detect by in vivo assays due to the mildness of their actions. The separation and extraction of some compounds could also be used in the industry in the way that tyrosol and other pharmaceutical products have also been studied.


THE FUTURE

An interesting, relatively new proposal was suggested by Israelidis (1990) for the treatment of bioremediation of alpechin.

The method (in combination with a novel membrane developed at the Research Centre KFA Julich of Germany in collaboration with the firm ATP Ltd, Iraklion of Greece) offers the following possibilities: (1) saving of water; and (2) recovery and production of valuable substances contained in the alpechin such as: (a) fertilisers between 65 and 80 kg/m3 of alpechin including organic and inorganic substances; (b) residual oil between 4 and 30 kg/m3 of alpechin; (c) colour substances (anthocyanines) 2-3 kg/m3 of alpechin; (d) pullulan (polysaccharide) and/or ethanol in quantities of 8 kg/m3 and 13 kg/m3 of alpechin, respectively.

This process had been primarily developed for sea water desalination, but it was then trial-tested to treat alpechin in Heraklion, Crete, and it appears to be very promising.

The point of issue is to develop an ecological and economical way to dispose of olive oil production waste-waters, with a simultaneous recovery of the water and of the valuable substances contained in the alpechin (residual oil, fertiliser, chemical compounds, fermentation by-products, etc.). The recovery and production of useful substances should be done using physicochemical and biotechnological methods.

These aims can be achieved with methods which detoxify alpechin and separate the water and the valuable substances from alpechin in order to obtain total recycling (excluding the residual oil).

Our own proposal is also based on the simultaneous recovery of the water and the valuable substances contained in alpechin. We suggest its use as composted fertiliser to extract chemical compounds that could be applied in the biopharmaceutical industry, and finally use of its biotransformation by-products such as the biopolymers.

Although not completely, we have reviewed here the most relevant applied systems and some considerations for future prospects.

We would like to point out that, although solutions are already available to dispose of alpechin, research efforts are still required to exploit the additional potential values available from alpechin.

ACKNOWLEDGEMENTS

The authors wish to thank the organisers for the opportunity to contribute to this Symposium. Research in the authors laboratory was supported


partly by the Spanish Ministry (project AMB 92 723 CO3-01) and the CEE (project EVWA-CT920006).

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