Journal of Biotechnology 98 (2002) 113 /123 www.elsevier.

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Environmental biotechnology: the ongoing quest

R. Grommen, W. Verstraete *
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Received 16 July 2001; accepted 16 January 2002

Abstract Environmental biotechnology, until now, has primarily focused on the development of technologies to treat aqueous, solid and gaseous wastes. At present, the basic knowledge on how biotechnology can handle these wastes has been acquired and the focus is now on the implementation of these processes as best available technology not entailing excessive costs (BATNEEC) in the framework of strict and transparent environmental legislation. New environmental challenges continue to evolve, as it becomes clear that waste streams should be tackled in an overall holistic way. New technologies to reach this goal are currently under development. Novel aspects with respect to the domain of water treatment are, for example, the biomembrane reactor technology and the newly discovered processes to remove nitrogen by means of anaerobic ammonium oxidation. Also, most challenging is the continuing strive for re-use of treated wastewater. Indeed, water shortage is emerging in an increasing number of countries all over the world and necessitates the short cycling of water. Finally, biotechnology has a key role to play in the novel approaches to design wastewater treatment based on decentralised sanitation and reuse (DESAR). Solid waste is a major challenge worldwide. The implementation of anaerobic digestion to treat biowastes has become a grown-up technology. New approaches in which biotechnological processes are linked to physical processes, such as plasma technology, certainly deserve special attention for the coming decades. Soil and sediment clean up by means of biostimulation/remediation/ augmentation is now well established. Certainly, a number of prospects need to be further explored, such as the use of special energy sources to stimulate in situ the microbial community and the seeding of knowledge to the in situ community by means of horizontal gene transfer mechanisms. A number of waste gases can be handled by biofilter systems. Biological treatment of wastegases is also evolving, inasmuch as that besides conventional chemical pollutants, now also highly problematic chemicals (even dioxins) can be dealt with through proper biotechnological approaches. A remarkable new potential is the use of well designed probiotics to upgrade aquaculture and together with conventional biological water treatment processes, to guarantee the overall water quality of this domain of food production. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biotechnology; Wastewater; Solid waste; Aquaculture

1. Framework for environmental technology and biotechnology

* Corresponding author. Tel.: '/32-9-264-5976; fax: '/32-9264-6248 E-mail address: willy.verstraete@rug.ac.be (W. Verstraete).

Environmental issues have been on the agenda for several decades now and it might therefore

0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 0 9 0 - 1

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seem that the hype of environmental technology is over. However, it cannot be denied that several environmental challenges continue to exist and their magnitude even increases. For instance, the spread of unwanted biological propagules is of major concern in Europe. The costs of remediating the spread of a prion, such as BSE or a virus, such as Foot and Mouth disease, costs the EU staggering amounts of money; for example, the direct economic impact of the BSE crises is estimated to cost each inhabitant some 13 Euro per year. The same applies to unwanted chemicals, recently referred to as POPs, i.e. persistent organic pollutants. The Belgian dioxin crisis was a severe lesson: it learned that PCBs and similar chemicals are all over the environment and particularly in fish products (Bernard et al., 1999). Currently, the residues of pharmaceuticals are of growing concern (Kummerer, 2001). Indeed, we use these very valuable chemicals, but we excrete them almost entirely in urine. The fate of pharmaceuticals in the environment is hardly studied. The omnipresence in the environment of ethynyl estradiol (EE2), the major component of the anticonception pill, is a perfect example of this (Kozak et al., 2001). Finally, to complete the list, the enrichment of the environment with unwanted inorganics, such as species of nitrogen (ammonia, nitrate), phosphorous (phosphate, phosphonates) and carbon (CO2; CO. . .), has not been curbed yet. We still need to draft the mass balances and the concomitant fluxes of the resources we use and the wastes we produce. Until now, environmental technology, and with it environmental biotechnology, has by no means been designed in a holistic way. Strangely enough, farming in general and farmland in particular is the crucial endpoint of many waste treatment processes. At the down-stream side of the wastewater treatment plant, organic fertiliser or even animal feed appears too often as a miraculous solution. The EU crisis in terms of food chain quality and consumer confidence, as experienced in the past, has resulted in the agroindustry coming under rigorous surveillance from soil to consumer and back to soil. Hazard analysis and critical control points (HACCP) will

be the key and all intermediate steps of the food chain will have to become traceable and accountable. The term sustainability is by now generally accepted. Yet, we have hardly set targets. One of the first cycles that we have to close is water. In the next decade, the shortage of water is predicted to rise considerably and we should learn to use the cycle of water in a more efficient way (e.g. by not using natural water systems as a point of dilution for waste products). Clearly, technology and particularly biotechnology have a major role to play in making water systems fully closed, both at the high-tech centralised and the low-tech decentralised level. It is evident that this generates a number of risks in terms of chemical and biological potentially dangerous propagules. A crucial element in the overall framework is the legislation and its implementation. Environmental quality depends on a well-designed set of international and national laws and regulations. In our currently worldwide entirely market-driven economy, it is very difficult to have such legislation accepted and even more difficult to have it implemented. We all know the dreadful story of the Kyoto agreements. Fortunately, within the EU, a strong commitment towards environmental quality has been demonstrated over the last decades, although the implementation of the various directives is another matter. Actually, the percentage of environmental directives not respected properly was recently published as follows: top countries in trespassing were Germany, Belgium and Spain with 9/10% not respected directives. If political associations with strong economies such as the US or the EU do not behave properly, how can we then expect any effort from third world countries. Clearly, the legal framework is a prerequisite for proper environmental and technical development. In this paper, a summary is given of the status of biotechnology in the different fields of action. Special attention is given to new ideas and promising evolutions are pointed out. Finally, a number of so-called super challenges are discussed.

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2. Key features of biotech for the environment The fact that microbial biocatalysis is relatively inexpensive is the predominant reason why so many environmental technologies prefer to rely on microorganisms. As a comparison: 1 kg waste (dry matter) costs in terms of incineration of the order of 0.5 Euro; biological mineralisation costs generally a factor 10 less. The second major advantage of microbiological processes is that they are flexible; they adapt to variable conditions (self-regulation) and also to new molecules or combinations of new molecules. A nice example is the fact that in recent years the recalcitrant herbicide atrazine has, at several locations, been found to be degraded by microorganisms (Yanzekontchou and Gschwind, 1994; Mandelbaum et al., 1995; Radosevich et al., 1995). Clearly, microbial species and communities continue to learn to metabolise or co-metabolise various xenobiotics. A third aspect is the fact that environmental biotech is perceived as green. We have all experienced that conventional sewage treatment installations with their predominance of concrete and electromechanics are not very much liked by the public, while reed beds and wetlands are considered environmentally useful. The message that the latter are quite expensive and can often not perform the treatment in a sufficient way is difficult to convey to the public. In our experience, the above mentioned features govern the continuous growing interest in environmental biotechnology.

Fig. 1. Evolution in world water use per sector (Shiklomanov, 1999).

3. The domains of action Considering the fact that environmental biotechnology can be operated in an inexpensive, flexible and also environmentally friendly way, there are a series of areas where microbial biocatalysis can and will expand in the near future. 3.1. Drinking water production The use of water is still rising exponentially, as shown in Fig. 1. In particular, the need for water

for crop production is remarkable. More and more countries become water scarce, this means that they do not have some 2000 m3 of fresh water per capita per year available. Table 1 shows a list of countries that will face water scarcity by 2025 (Seckler et al., 1999). Note that the Peoples Republic of China and India are also on the list. These countries have severe regional water scarcity, only portions of their populations will face absolute water scarcity. Parallel with the water shortage goes the fact that the public distrusts more and more public drinking water supply. In the EU, the use of bottled water increases 5% per year. It should be noted that this means that more and more people are willing to pay in the order of 0.5 Euro per litre. The reasons for the public concern are manifold. There is the presence of unwanted organisms such as Cryptosporidium, Legionella, . . . There is the occurrence of trace chemicals, such as trihalomethanes, nonylphenol, EE2 and other putative estrogens, the presence of pesticides and residual pharmaceuticals. . . Biotechnology has a major contribution to make in this field. First of all, by means of specific bioassays, one can monitor for active chemicals such as estrogens (Routledge and Sumpter, 1996). Secondly, one can now, by means of molecular methods such as denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridisation (FISH), rapidly screen for microbial species (Dewettinck et al., 2001a). In the coming

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Table 1 List of countries that will experience water scarcity by 2025 Category 1 Afghanistan Egypt Iran Iraq Israel Jordan Kuwait Libya Oman Pakistan Saudi Arabia Singapore South Africa Syria Tunisia United Arab Emirates Yemen (Peoples Republic of China)a (India)a Category 2 Angola Benin Botswana Burkina Faso Burundi Cameroon Chad Congo Cote dIvoire Ethiopia Gabon Ghana Guinea-Bissau Haiti Lesotho Liberia Mozambique Niger Nigeria Paraguay Somalia Sudan Uganda D.R. Congo

Category 1 lists countries with absolute water scarcity. They will not be able to meet water needs in the year 2025. Category 2 lists countries with economic scarcity. They will have to more than double their efforts to extract water to meet 2025 water needs, but they will not have the financial resources available to develop these water supplies. a These countries have severe regional water scarcity. Only a portion of their populations (381 million people in Peoples Republic of China and 280 million people in India based on estimates of 1990) is in Category 1 (Seckler et al., 1999).

1993). These microbial processes have the major advantage over conventional granular activated carbon (GAC) that they can decrease, besides the apolar pollutants, also the polar contaminants. The major question to be answered however is how far down the microorganisms are capable of removing the chemicals of concern because, in the case of estrogens, ng l (1 levels are required. A special feature in the domain of drinking water is the in-house extra treatment facility. Indeed, several companies have launched systems on the consumer market to add to the tap and thus to further treat tap water. These systems not only have to improve the water, they must also remain free of microbial contamination. Progress so far appears to warrant optimism. A special issue is the re-use of treated water as drinking water. At the industrial level, it appears quite possible to provide a well designed loop in which the quality of the water is controlled according to the HACCP concept (Dewettinck et al., 2001b). Fig. 2 shows a scheme illustrating the use of the HACCP concept to guarantee safe drinking water production. Several countries have now launched projects in which at the level of the single house or housing complex, water is treated and to some extent re-used. It remains a major challenge to design a small-scale loop to produce drinking water from, for example, grey water. Yet, the need is growing all over the world. 3.2. Aerobic wastewater treatment The facts about the current aerobic technology to treat wastewater are that it generally occurs in a centralised way that is effective at first sight. A closer look reveals that such treatment is still poorly understood: it relies on microbial communities which come to existence but, as yet, are hard to control by, for example, the addition of certain supplements (Vansever et al., 1997; Seka et al., 2001). Moreover, the conventional treatment is costly, i.e. :/100 Euro per inhabitant per year. Another major drawback is the fact that it is not holistic at all: half of the organic matter (COD) becomes sludge which has to be disposed of either by depositing it onto the soil or by incineration. Most of the nitrogen becomes N2 or N2O and is

decade, a set of commercial molecular microarrays will be available for online safeguarding of the quality of drinking and process water. Biotechnology has also a unique role to play in the technology of drinking water production. Combined physical and biological process are capable of removing pollutants down to the mg l (1 level. Madoni et al. (2001) described a combination of nitrifiers with manganese oxidising bacteria. Several approaches to implement Fe(0) oxidising Shewanella to remove organochlorines are underway. Microbial removal of nitrate by methanol is applied on a full scale (Liessens et al.,

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Fig. 2. Illustration of the use of the HACCP concept to guarantee the production of safe drinking water. (POA, point of attention; CCP, critical control point; QRA, quantitative risk assessment; vir, viruses; prot, protozoa; RO, reverse osmosis; MF, micro-ltration) (after Dewettinck et al., 2001a).

lost. There is, at present, no technology available to re-use the P and finally the real end product of the treatment is called effluent because it rarely qualifies as water. Combined sewerage has the tremendous advantage of exporting the putative infectious material outside the cities. However, it prevents the development of niche technologies for recovery of potential valuable compounds (e.g. as is proposed by the DESAR principle, see further). The vested interests of mega-companies can have as a result that, for many years to come, the conventional aerobic wastewater treatment approach will be forced on sites and in situations (particularly in third world countries) where it is not appropriate. In terms of new approaches, two lines of action are certainly emerging. First there is the growing interest in source separation in which rain water, grey water (bath, kitchen. . .) and black water (fecal matter) are separated. Each can then be subsequently treated in an appropriate way (Larsen and Gujer, 1996). Secondly, coherent processes

such as biomembrane reactors with minimal sludge production respectively nitrification /denitrification without need of organic carbon are under development. With respect to the processes of nitrogen removal, really remarkable novel discoveries in the field of microbial physiology are occurring, such as the sulphate driven ammonium oxidation process (Fdz-Polanco et al., 2001), the anaerobic ammonium oxidation process (Mulder et al., 1995) and the so-called oxygen limited nitrification and denitrification process (OLAND) in which conventional nitrifiers perform the N2 production (Kuai and Verstraete, 1998). The new overall philosophy for aerobic wastewater treatment is coined in the term DESAR, decentralised sanitation and re-use. Fig. 3 schematises the ideal situation in which the niche technologies are infiltration of rainwater, digestion and use as fertiliser of the fecal matter and extensive treatment of the grey water. It should be noted that this scheme allows local production

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Fig. 3. Scheme of the DESAR principle.

of drinking water by means of the replenished ground water table and also the use of so-called ecological water (e-water) for activities such as washing and rinsing. A central element in the concept is that the citizen will be responsible for his own safety and health. Obviously, we need to develop a set of simple robust control systems to ascertain that DESAR is one step forward in terms of a sustainable water technology. 3.3. Anaerobic wastewater treatment (Verstraete and Vandevivere, 1999) There is little doubt that anaerobic digestion of wastewater is a mature technology. Worldwide, several hundreds of reactors have been built for industrial wastewaters. They treat these waters with high efficiency, producing hardly any excess sludge and at an overall cost which is a factor two to three lower than the aerobic counterpart. The major potential at this moment for the anaerobic digestion is the fact that the biogas can be considered as green energy. As a matter of fact,

1 kg COD equals :/0.5 m3 of biogas, which in turn yields the equivalent of 0.1 Euro of electricity. A growing number of European countries are currently in the framework of the Kyoto agreements, granting green energy certificates to biogas, which certainly boosts the interests in this technology. At the level of the biotechnology in the strict sense, there is a need for more progress in the hydrolysis/digestion of particulate organic matter. There are currently interesting developments with respect to chemical enhanced precipitation treatment (Kalogo and Verstraete, 2000). Also chemical/physical treatments of the particulates to render them more fermentable, such as ozonation (Weemaes et al., 2000), grinding (Weemaes and Verstraete, 1998) and even subcritical gasification (Misch et al., 2000) are emerging. Of course, there is also the onset of anaerobic biomembrane reactor technology to be expected. Anaerobic digestion performs poorly at low temperatures and therefore needs to be linked to low-value heat recovery processes. However, for

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instance, for domestic sewage, at the current prices of aerobic sludge treatment it might become more feasible in a number of situations to opt for the total costs of heating up the sewage and treating it directly in an anaerobic way. As a matter of fact, the heating costs of some 25 Euro per inhabitant per year might balance the growing costs to handle and dispose the excess sludge that would be generated by an aerobic treatment. Finally, in most instances, anaerobic wastewater treatment is mainly focused on organic carbon removal and needs to be completed by an adequate after-treatment for N, P and infectious propagules. Possible candidates are certainly the above mentioned anaerobic ammonium oxidation processes and emerging electrochemical advanced post-treatments (Wang et al., 2001).

Fig. 4. Proposed scheme for the treatment of grey wastes.

3.5. Soil and sediment clean up 3.4. Solid waste handling Only a decade ago, biotechnology appeared to have lost its role in handling solid wastes. Indeed, aerobic composting had to struggle with the large fraction of non-biodegradables and delivered a low-value product. At present, the role of biotech in solid waste handling is growing steadily. The basis for this is source-separated collection. Separation of the wet organics at home allows the treatment of the latter in a convenient way and also to produce a dry grey fraction that is better suited for combustion. As a whole, anaerobic digestion of biowastes is steadily growing into a fully matured technology (Vandevivere and Verstraete, 2001). Even the fermentative treatment of grey wastes is currently examined because it permits the bringing of the recovery of certain valuable materials into consideration. Some additional developments in this domain are: the design of apparatus (and matrixes plus inocula) for inhouse kitchen composters, the development of low-tech concepts for anaerobic digestion coupled to refuse-derived fuel production for third world countries (Biey and Verstraete, 1999) and the integration of digestion with incineration, respectively, plasma treatment for total waste handling at a megascale (Fig. 4). The last 5 years have seen a renaissance of soil microbiology in general and soil microbial ecology in particular. Numerous new processes and organisms are described for the deep soil (Marchesi et al., 2001). As to surface soils, there is a formidable interest in clean organic farming and methods to ascertain by means of microbiological assays that this practice is implemented for specific areas of agricultural production (Carpenter-Boggs et al., 2000). Moreover, there is a tremendous interest in bioremediation and so-called natural attenuation of soils and sediments. The recent advances in molecular ecology have been breathtaking and have provided remarkably efficient ways to certify genomic diversity and richness of soils and sediments. These developments are now powerful tools to evaluate the effect of pesticides on the structure and diversity of the soil microbial community (Engelen et al., 1998) and to compare polluted sites and bioremediated sites (Boon et al., 2000). This is a major step forward. Similarly, there have been most interesting reports on how to bring about information (genes) into existing microbial communities of soils and sediments by the mechanism of horizontal gene transfer (van Elsas et al., 2000). This approach,

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combined with the infusion of a selective energy source, such as for example methane gas, opens perspectives to activate powerful microbial consortia not only in surface soils, but also in sediments and deep soils. The problems in this domain of environmental biotechnology are that one has, thus far, very few handles to measure/control the much praised microbial diversity and homeostasis. It is clear that the heterogeneity of the soil is key to the microbial bio-diversity. At present, the most powerful approach to support microbial diversity in soils and sediments is to provide specific plant roots (so-called rhizoremediation). Obviously, a great area of exploration in which micro-organisms in cooperation or even symbiosis (mycorrhiza) with plants are used to treat inorganic and organic pollutants in the soil is at bay (Meharg and Cairney, 2000). A final remark concerns the fact that, with respect to soils and sediments, the rate of action is still far too slow. Too much focus on natural attenuation is nothing but deliberate postponement. It is imperative that legislation becomes more rigorous and activates mechanisms that make the use of soil, respectively, its transfer in ownership directly coupled to underwriting a project to clean up the soil matrix by means of the best available technology, in a well defined period. Such legislative actions have become effective in some EU countries and certainly deserve to be extended. 3.6. Off gases and air purication The contribution of biotechnological processes to the treatment of polluted gases is particularly documented for the so-called biofilter systems (Muthumbi et al., 2000). Biofilter technology is excellent for low-level volatile organic compounds (VOCs); it should also be noted that bioaugmentation can effectively be applied for these systems (Smet et al., 1996). A quite remarkable development in this field is the removal of H2S and its recovery as S by the Thiobacillus process (Buisman et al., 1990). This technology has further evolved into full-scale procedures to bring about flue gas desulfurisation

(Lens and Hulshoff Pol, 2000). Clearly, there is quite a potential along these lines. The major challenges with respect to gases and air are, however, the so-called greenhouse gases and the dioxins. For the latter, which are very much feared in the vicinity of garbage incinerators, biotech has some potential inasmuch as it can provide very sensitive bio-assays to detect, for instance, estrogenic activity of aryl hydrocarbon activity (Muthumbi et al., 2002). Yet, biotech can also become a major player in the decrease of gases contributing to the greenhouse effect. First of all, methanotrophic bacteria have a crucial role to play in the caption of atmospheric methane and particularly in the methane emitted from landfills. Design of proper cover layers effectively treating the methane diffusing form such landfills does not appear impossible (Boeckx and Van Cleemput, 1996). Secondly, if one considers the global cycling of carbon, it is apparent that one major process to be considered is the mineralisation of the soil organic matter pool by the microbiota. The development of a procedure to decrease that mineralisation rate, e.g. by water logging or a process to slow down the breakdown of compost, respectively, soil humus, e.g. by increasing the cross linking of the molecules, could give considerable new perspectives to the current concerns of the dwindling reserves of carbon captured in soils and sediments. 3.7. Aquaculture technology The increase of the world food fish production, which was estimated at 20 million tons over the last decade, can be mainly attributed to aquaculture. Indeed, while the total catch from fisheries has been levelling off since the last decade, due to the fact that the majority of the stocks are being fully exploited, aquaculture has been growing constantly at a rate of 10% per year (FAO, 2000). Waste discharges of aquaculture facilities can have a pronounced effect on their surroundings (Wu, 1995). Increased sedimentation of organic material below fish cages, nutrient release in receiving waters and the use of antibiotics are a few examples of possible negative impacts of aquaculture on the environment.

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New technologies, such as intensive recirculation systems, are currently being explored (Ebeling, 2000). These systems include solids removal, nitrification of ammonia and nitrite and oxygenation of the recirculating water. One of the key processes in these water treatment systems is the removal of toxic ammonia by nitrification. New methods to incorporate nitrifying bacteria in socalled biofilters have been proposed (Kim et al., 2000). Other research focuses on the use of nitrifying inocula to accelerate the start-up of a biofilter (Grommen et al., 2002) as nitrifying bacteria have very low growth rates. Another important issue is the use of probiotics to improve the fish health and to increase growth rates (Verschuere et al., 2000), as it could be an alternative to the excessive use of antibiotics. Especially in the field of larviculture, the use of probiotics could prove a long-awaited improvement, as it is assumed that the uncontrolled development of the microbial communities in hatcheries is one of the major reasons for the unpredictable and often variable results. Recently, Nikoskelainen et al. (2001) investigated the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, as a probiotic for fish. The strains were specifically chosen as they are known to be safe for humans.

4. The super challenges Overall, environmental technology and environmental biotechnology have some major contributions to make in the years to come. First of all, we need to document and model in much more detail the existence, survival and potential spread of biological propagules (protozoa, bacteria, viruses and prions) in the environment. At present, there is just too much uncertainty to live and deal comfortably with them. The same goes for a number of chemicals that the natural microorganisms appear not to act upon readily, the so-called POPs. We should document their dynamics more precisely. As indicated, the drinking water quality must be considerably increased. This may require processes that only handle the few litres of water per person

per day really devoted to consumption. These processes however will have to be able to remove pollutants down to the ng l (1 level. In the aerobic wastewater treatment, new approaches are needed in which the three-dimensional positioning of the microorganisms in the microbial consortia are better understood and can be engineered. Moreover, attention should be given to food chain systems in which bacteria are consumed by protozoa, the latter by daphnia etc., thus giving rise to low surplus sludge production. Most of all, the concepts of decentralised treatment units and their potential to generate re-uses must be explored and scrutinised. In the anaerobic wastewater treatment, priority must be given to direct anaerobic treatment of domestic sewage, particularly for third world countries. This will allow the decrease of investment and operation costs of water treatment. Solid wastes offer the potential to give anaerobic biotech a place in the world of green energy production. Indeed, the Kyoto agreements stipulate that industrial countries should obtain some 12.5% of their electricity via renewable resources. By anaerobic digestion, there is a potential stake for biotechnologists. Soils and sediments are the beholders of the microbial diversity and therefore deserve to be preserved with utmost care. Their intrinsic heterogeneity is key to their richness in species and needs, for example by means of proper rhizotechnology, to be further developed. Moreover, horizontal gene transfer technology allows the engineering of effective in situ soil communities and processes. Concerning the production of greenhouse gases, it is believed that soil biotech can have a major task in terms of slowing down the organic carbon mineralisation and thus contributing to the concept of carbon sequestration. Finally, as agriculture is shifting in many ways and aquaculture in particularly is booming, it appears that environmental biotechnology in this domain can give an important contribution, for example by formulating effective probiotics, to rear and produce healthy fish in high quality water.

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