Microorganisms

Biodegradation will be described associated with environmental bioremediation. Therefore, biodegradation is nature’s way of recycling wastes, or breaking down organic matter into nutrients that can be used and reused by other organisms. In the microbiological sense, “biodegradation” means that the decaying of all organic materials is carried out by a huge assortment of life forms comprising mainly bacteria, yeast and fungi, and possibly other organisms.

Bioremediation and biotransformation methods endeavour to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), radionuclides and metals.

Figure : Microbial processes used in bioremediation technologies.

Some biodegradable pollutants

In the last few decades, highly toxic organic compounds have been synthesized and released into the environment for direct or indirect application over a long period of time. Fuels, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), pesticides and dyes are some of these types of compounds. Some other synthetic chemicals like radionuclides and metals are extremely resistant to biodegradation by native flora compared with the naturally occurring organic compounds that are readily degraded upon introduction into the environment.

Hydrocarbons: are organic compounds whose structures consist of hydrogen and carbon. Hydrocarbons can be seen as linear linked, branched or cyclic molecules. They are observed as aromatic or aliphatic hydrocarbons. The first one has benzene (C₆H₆) in its structure, while the aliphatic one is seen in three forms: alkanes, alkenes and alkynes

Polycyclic aromatic hydrocarbons (PAHs): are important pollutants class of hydrophobic organic contaminants (HOCs) widely found in air, soil and sediments. The major source of PAH pollution is industrial production. They have been studied with increasing interest for more than twenty years because of more findings about their toxicity, environmental persistence and prevalence. PAHs can sorb to organic-rich soils and sediments, accumulate in fish and other aquatic organisms, and may be transferred to humans through seafood consumption. The biodegradation of PAHs can be considered on one hand to be part of the normal processes of the carbon cycle, and on the other as the removal of man-made pollutants from the environment. The use of microorganisms for bioremediation of PAH-contaminated environments seems to be an attractive technology for restoration of polluted sites.

Polychlorinated biphenyls (PCBs): are mixtures of synthetic organic chemicals. Due to their non-flammability, chemical stability, high boiling point, and electrical insulating properties, PCBs were used in hundreds of industrial and commercial applications including electrical, heat transfer, and hydraulic equipment; as plasticizers in paints, plastics, and rubber products; in pigments, dyes, and carbonless copy paper; and many other industrial applications. Consequently, PCBs are toxic compounds that could act as endocrine disrupters and cause cancer. Therefore, environmental pollution with PCBs is of increasing concern.

Pesticides: are substances or mixture of substances intended for preventing, destroying, repelling or mitigating any pest. Pesticides which are rapidly degraded are called nonpersistent while those which resist degradation are termed persistent. The most common type of degradation is carried out in the soil by microorganisms, especially fungi and bacteria that use pesticides as food source.

Dyes: are widely used in the textile, rubber product, paper, printing, color photography, pharmaceuticals, cosetics and many other industries. Azo dyes, which are aromatic compounds with one or more (–N=N–) groups, are the most important and largest class of synthetic dyes used in commercial applications. These dyes are poorly biodegrabale because of their structures and treatment of wastewater containing dyes usually involves physical and / or chemical methods such as adsorption, coagulation-flocculation, oxidation, filtration and electrochemical methods.

Radionuclides: a radionuclide is an atom with an unstable nucleus, characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or via internal conversion. During this process, the radionuclide is said to undergo radioactive decay, resulting in the emission of gamma ray(s) and/or subatomic particles such as alpha or beta particles.

Heavy metals: unlike organic contaminants, the metals cannot be destroyed, but must either be converted to a stable form or removed. Bioremediation of metals is achieved through biotransformation. Mechanisms by which microorganisms act on heavy metals include biosorption (metal sorption to cell surface by physicochemical mechanisms), bioleaching (heavy metal mobilization through the excretion of organic acids or methylation reactions), biomineralization (heavy metal immobilization through the formation of insoluble sulfides or polymeric complexes), intracellular accumulation, and enzyme-catalyzed transformation (redox reactions). The major microbial processes that influence the bioremediation of metals are summarized in Figure above.

Bacterial degradation

There are many reports on the degradation of environmental pollutants by different bacteria. Several bacteria are even known to feed exclusively on hydrocarbons. Bacteria with the ability to degrade hydrocarbons are named hydrocarbon-degrading bacteria. Biodegradation of hydrocarbons can occur under aerobic and anaerobic conditions, it is the case for the nitrate reducing bacterial strains Pseudomonas sp. and Brevibacillus sp. isolated from petroleum contaminated soil. However, data presented by Wiedemeier et al. suggest that the anaerobic biodegradation may be much more important. 25 genera of hydrocarbon degrading bacteria were isolated from marine environment. Furthermore, among 80 bacterial strains isolated by Kafilzadeh et al. which belonged to 10 genus as follows: Bacillus, Corynebacterium, Staphylococcus, Streptococcus, Shigella, Alcaligenes, Acinetobacter, Escherichia, Klebsiella and Enterobacter, Bacillus was the best hydrocarbon degrading bacteria.

Bacterial strains that are able to degrade aromatic hydrocarbons have been repeatedly isolated, mainly from soil. These are usually gram negative bacteria, most of them belong to the genus Pseudomonas. The biodegradative pathways have also been reported in bacteria from the genera Mycobacterium, Corynebacterium, Aeromonas, Rhodococcus and Bacillus.

Although many bacteria are able to metabolize organic pollutants, a single bacterium does not possess the enzymatic capability to degrade all or even most of the organic compounds in a polluted soil. Mixed microbial communities have the most powerful biodegradative potential because the genetic information of more than one organism is necessary to degrade the complex mixtures of organic compounds present in contaminated areas.

Both, anaerobic and aerobic bacteria are capable of biotransforming PCBs. Higher chlorinated PCBs are subjected to reductive dehalogenation by anaerobic microorganisms. Lower chlorinated biphenyls are oxidized by aerobic bacteria. Research on aerobic bacteria isolated so far has mainly focused on Gram-negative strains belonging to the genera Pseudomonas, Burkholderia, Ralstonia, Achromobacter, Sphingomonas and Comamonas. However, several reports about PCB-degrading activity and characterization of the genes that are involved in PCB degradation indicated PCB-degrading potential of some Gram-positive strains as well (genera Rhodococcus, Janibacter, Bacillus, Paenibacillus and Microbacterium). Aerobic catabolic pathway for PCB degradation seems to be very similar for most of the bacteria and comprises four steps catalysed by the enzymes, biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2, 3-dihydroxybihenyl dioxygenase (DHBD) (BphC) and hydrolase (BphD).

Researches on bacterial strains that are able to degrade azo dyes under aerobic and anaerobic conditions have been extensively reported. Based on the available literature, it can be concluded that the microbial decolourization of azo dyes is more effective under anaerobic conditions. On the other hand, these conditions lead to aromatic amine formation, and these are mutagenic and toxic to humans requiring a subsequent oxidative (aerobic) stage for their degradation. In this context, the combined anaerobic/aerobic biological treatments of textile dye effluents using microbial consortia are common in the literature. For exemple, Chaube et al. have used the mix consortia of bacteria consisting of Proteus sp., Pseudomonas sp. and Enterococcus sp. in biodegradation and decolorisation of dye. However, several researchers have identified single bacterial strains that have very high efficacy for removal of azo dyes, it is the case of Shewanella decolorations. In contrast to mixed cultures, the use of a pure culture has several advantages. These include predictable performance and detailed knowledge on the degradation pathways with improved assurance that catabolism of the dyes will lead to nontoxic end products under a given set of environmental conditions. Another advantage is that the bacterial strains and their activity can be monitored using culture-based or molecular methods to quantify population densities of the bacteria over time. Knowledge of the population density can be extrapolated to quantitative analysis of the kinetics of azo dye decoloration and mineralization.

Heavy metals cannot be destroyed biologically (no“degradation”, change in the nuclear structure of the element, occurs) but are only transformed from one oxidation state or organic complex to another. Besides, bacteria are also efficient in heavy metals bioremediation. Microorganisms have developed the capabilities to protect themselves from heavy metal toxicity by various mechanisms, such as adsorption, uptake, methylation, oxidation and reduction. Reduction of metals can occur through dissimilatory metal reduction, where bacteria utilize metals as terminal electron acceptors for anaerobic respiration. In addition, bacteria may possess reduction mechanisms that are not coupled to respiration, but instead are thought to impart metal resistance. For example, reduction of Cr(VI) to Cr(III) under aerobic or anaerobic conditions, reduction of Se(VI) to elemental Se, reduction of U(VI) to U(IV) and reduction of Hg(II) to Hg(0). Microbial methylation plays an important role in heavy metals bioremediation, because methylated compounds are frequently volatile. For example, Mercury, Hg(II) can be biomethylated by a number of different bacterial species Alcaligenes faecalis, Bacillus pumilus, Bacillus sp., P. aeruginosa and Brevibacterium iodinium to gaseous methyl mercury. In addition to redox conversions and methylation reactions, acidophilic iron bacteria like Acidithiobacillus ferrooxidans and sulfur oxidizing bacteria are able to leach high concentrations of As, Cd, Cu, Co and Zn from contaminated soils. On the other hand metals can be precipitated as insoluble sulfides indirectly by the metabolic activity of sulphate reducing bacteria. Sulphate reducing bacteria are anaerobic heterotrophs utilizing a range of organic substrates with SO₄ – as the terminal electron acceptor. Heavy metal ions can be entrapped in the cellular structure and subsequently biosorbed onto the binding sites present in the cellular structure. This method of uptake is independent of the biological metabolic cycle and is known as biosorption or passive uptake. The heavy metal can also pass into the cell across the cell membrane through the cell metabolic cycle. This mode of metal uptake is referred as active uptake. Pseudomonas strain, characterized as part of a project to develop a biosorbent for removal of toxic radionuclides from nuclear waste streams, was a potent accumulator of uranium (VI) and thorium (IV).

Most works on pollutants bioremediation uses pure microbial cultures. However, the use of mixed microbial cultures is undoubtedly advantageous. Some of the best examples of enrichment cultures comprising several specific consortia involve the bioremediation. In the case of heavy metals removal, Adarsh et al. have used an environmental bacterial consortium to remove Cd, Cr, Cu, Ni and Pb from a synthetic wastewater effluent. For Cr(VI) removal we reported that the survival and stability of bacteria are better when they are present as a mixed culture, especially, in highly contaminated areas and in the presence of more than one type of metal. Indeed, the indigenous bacteria enriched from chromium contaminated biotopes, were able to remove Cr(VI) successfully in multi-contaminated heavy metal solution. A microbial consortium consisting of three bacterial Pseudomonas species originally obtained from dye contaminated sites was capable of decolorizing textile effluent and dye faster than the individual bacteria under static conditions.
PGPR and PGPB degradation

Plant associated bacteria, such as endophytic bacteria (non-pathogenic bacteria that occur naturally in plants) and rhizospheric bacteria (bacteria that live on and near the roots of plants), have been shown to contribute to biodegradation of toxic organic compounds in contaminated soil and could have potential for improving phytoremediation. Plant growth promoting rhizobacteria (PGPR) are naturally occurring soil bacteria that aggressively colonize plant roots and benefit plants by providing growth promotion. Some plants can release structural analogs of PAHs such as phenols, to promote the growth of hydrocarbon degrading microbes and their degradation on PAHs. For such plant/microbe systems, an important class of bacteria is Pseudomonas spp., have PGPR activity and hydrocarbon degrading capacity. Furthermore, the rhizosphere of vegetation in contaminated field contains higher diversity of population of PAH-degrading bacteria, among which two Lysini bacillus strains were isolated. Culturable PCB degraders were also associated with both the rhizosphere and root zone of mature trees growing naturally in a contaminated site, they were identified as members of the genus Rhodococcus, Luteibacter and Williamsia, which suggest that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ. Also, the free living nitrogen fixer Azospirillum lipoferum generally found in the rhizoplane of the crop plants was used for Malathion degradation which is one of the largest organo phosphorus insecticides in the world. Results from the literature suggest that heavy metals may be removed from contaminated soils using plant growth promoting rhizobacteria. The use of soil bacteria (often plant growth promoting bacteria (PGPB)) as adjuncts in metal phytoremediation can significantly facilitate the growth of plants in the presence of high (and otherwise inhibitory) levels of metals. To increase the efficiency of contaminants extraction, it is interesting to apply plants combined to some microorganisms; such technique is called rhizoremediation.

Microfungi and mycorrhiza degradation

Microfungi are described as a group of organisms that constitute an extremely important and interesting group of eukaryotic, aerobic microbes ranging from the unicellular yeasts to the extensively mycelial molds. Yeasts preferentially grow as single cells or form pseudomycelia, whereas molds typically grow as mycelia-forming real hyphae. Fungi are an important part of degrading microbiota because, like bacteria, they metabolize dissolved organic matter; they are principal organisms responsible for the decomposition of carbon in the biosphere. But, fungi, unlike bacteria, can grow in low moisture areas and in low pH solutions, which aids them in the breakdown of organic matter. Equipped with extracellular multienzyme complexes, fungi are most efficient, especially in breaking down the natural polymeric compounds. By means of their hyphal systems they are also able to colonize and penetrate substrates rapidly and to transport and redistribute nutrients within their mycelium. Mycorrhiza is a symbiotic association between a fungus and the roots of a vascular plant. In a mycorrhizal association, the fungus colonizes the host plant’s roots, either intracellularly as in arbuscular mycorrhizal fungi (AMF), or extracellularly as in ectomycorrhizal fungi. They are also an important component of soil life and soil chemistry. Bioremediation using mycorrhiza is named mycorrhizoremediation. Fungi possess important degradative capabilities that have implications for the recycling of recalcitrant polymers (e.g., lignin) and for the elimination of hazardous wastes from the environment. Below, some aspects of the microfungal degradation of some pollutants by unicellular and filamentous fungi are discussed.

Yeasts degradation

Several yeasts may utilize aromatic compounds as growth substrates, but more important is their ability to convert aromatic substances cometabolically. Some species such as the soil yeast Trichosporon cutaneum possess specific energy-dependent uptake systems for aromatic substrates (e.g., for phenol).

Furthermore, biodegradation of aliphatic hydrocarbons occurring in crude oil and petroleum products has been investigated well, especially for yeasts. The n-alkanes are the most widely and readily utilized hydrocarbons, with those between C10 and C20 being most suitable as substrates for microfungi. However, the biodegradation of n-alkanes having chain lengths up to n-C24 has also been demonstrated. Typical representatives of alkane-utilizing yeasts include Candida lipolytica, C. tropicalis, Rhodoturularubra, and Aureobasidion(Trichosporon) pullulans. Rhodotorula aurantiaca and C. ernobii were found able to degrade diesel oil. Yeasts are also reported for aniline biodegradation (a potential degradation product of the azo dye breakdown) it is the example of C. methanosorbosa BP-6. According to many authors, bacteria have been described as being more efficient hydrocarbon degraders than yeast, or at least that bacteria are more commonly used as a test microorganism. However, there is information that yeasts are better hydrocarbon degraders than bacteria.

In addition to aromatic and aliphatic hydrocarbons compounds, microfungi may transform numerous of other aromatic organopollutants cometabolically, including polycyclic aromatic hydrocarbons (PAHs) and biphenyls, dibenzofurans, nitro aromatics, various pesticides, and plasticizers. There have also been studies of PCB metabolism by yeasts C. boidinii and C. lipolytica and Saccharomyces cerevisiae. Insecticides and fungicides can also be adsorbed by S. cerevisiae during aerobic fermentation.

Yeasts are known for playing an important role in the removal of toxic heavy metals. There are many reports on biosorption of heavy metals by yeasts. Several investigations demonstrated that yeasts are capable of accumulating heavy metals such as Cu(II), Ni(II), Co(II), Cd(II) and Mg(II) and are superior metal accumulators compared to certain bacteria. In the case of hexavalent chromium (Cr(VI)) we found that P. anomala is able to remove Cr(VI) and we studied the biosorption of Cr(VI) by live and dead cells of three yeasts species: Cyberlindnera fabianii, Wickerhamomyces anomalus and C. tropicalis. Several yeast strains S. cerevisiae, P. guilliermondii, Rhodotorula pilimanae, Yarrowiali polytica and Hansenula polymorpha have been reported to reduce Cr(VI) to Cr(III). In addition, the tolerance of P. guilliermondii to chromate was found to depend on its capacity for extracellular reduction of Cr(VI) and Cr(III) chelation Most studies, have reported the efficiency of immobilized cells of yeasts in metals removal, one example is Schizosaccharomyces pombe for copper removal.
Filamentous fungi degradation

The attributes that distinguish filamentous fungi from other life forms determine why they are good biodegraders. First, the mycelial growth habit gives a competitive advantage over single cells such as bacteria and yeasts, especially with respect to the colonization of insoluble substrates. Fungi can rapidly ramify through substrates, literally digesting their way along by secreting a battery of extracellular degradative enzymes. Hyphal penetration provides a mechanical adjunct to the chemical breakdown affected by the secreted enzymes. The high surface to cell ratio characteristic of filaments maximizes both mechanical and enzymatic contact with the environment. Second, the extracellular nature of the degradative enzymes enables fungi to tolerate higher concentrations of toxic chemicals than would be possible if these compounds had to be brought into the cell. In addition, insoluble compounds that cannot cross a cell membrane are susceptible to attack.

Many workers divide bioremediation strategies into three general categories:

• the target compound is used as a carbon source;
• the target compound is enzymatically attacked but is not used as a carbon source (cometabolism) and
• the target compound is not metabolized at all but is taken up and concentrated within the organism (bioaccumulation).

Although fungi participate in all three strategies, they are often more proficient at cometabolism and bioaccumulation than at using xenobiotics as sole carbon sources. The isolates identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum are toluene-degrading fungi, they use toluene as sole carbon and energy source.

The majority of filamentous fungi are unable to totally mineralize aromatic hydrocarbons; they only transform them into indirect products of decreased toxicity and increased susceptibility to decomposition with the use of bacteria suggesting that the interaction among fungi and bacteria is profitable for the process of petroleum hydrocarbon mineralization. Among the filamentous fungi participating in aliphatic hydrocarbon biodegradation are Cladosporium and Aspergillus, whereas fungi belonging to Cunninghamella, Penicillinum, Fusarium and Aspergillus can take part in aromatic hydrocarbon decomposition. Fungal genera, namely, Amorphoteca, Neosartorya and Talaromyces were isolated from petroleum contaminated soil and proved to be the potential organisms for hydrocarbon degradation. A group of fungi, namely, Aspergillus, Cephalosporium and Pencillium was also found to be potential degrader of crude oil hydrocarbons. Fungal potentiality in PCBs degradation has been rarely explored. Several studies revealed that filamentous fungi can degrade PCBs. Among the filamentous fungi, the ligninolytic ones have been specifically investigated because of their extracellular, aspecific oxido-reductive enzymes that have been already successfully exploited in the degradation of many aromatic pollutants. There have also been studies of PCB metabolism by ectomycorrhizal fungi and other fungi such as Aspergillus niger. Fungi are also reported to degrade standing timber, finished wood products, fibers, and a wide range of non cellulosic products such as plastics, fuels, paints, glues, drugs, and other human artifacts. Fungi are known to tolerate and detoxify metals by several mechanisms including valence transformation, extra and intracellular precipitation and active uptake. Many species can adsorb cadmium, copper, lead, mercury, and zinc into their mycelium and spores. Sometimes the walls of dead fungi bind better than living ones. Systems using Rhiloprzs arrhizus have been developed for treating uranium and thorium. Aspergillus niger AB10 during cadmium and R. arrhizus M1 during lead biosorption indicated that the cell surface functional groups of the fungus might act as ligands for metal sequestration resulting in removal of the metals from the aqueous culture media. Furthermore, the proteins in the cell walls of AMF appear to have similar ability to sorb potentially toxic elements by sequestering them. There is evidence that AMF can withstand potentially toxic elements and glomalin produced on hyphae of AMF can sequester them. AMF plays a significant ecological role in the phytostabilization of potentially toxic trace element polluted soils by sequestration and, in turn, help mycorrhizal plants survive in polluted soils.

The most widely researched fungi in regard to dye degradation are the ligninolytic fungi. Nine strains of filamentous fungi were isolated by Abruscia et al. from cinematographic film consisted of three species of Aspergillus i.e. A. ustus, A. nidulans var. nidulans, A. versicolor, as well as, Penicillium chrysogenum, Cladosporium cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata and Trichoderma longibrachiatum were able to biodegrade the gelatin emulsion with different rates of metabolic CO₂ production. Filamentous fungi may degrade pesticides using two types of enzymatic systems: intracellular (cytochromes P450) and exocellular (lignin-degrading system mainly consisting in peroxidases and lactases). Each of these systems could also be induced or inhibited by pesticides, thus able to modulate their metabolism.

Degradative capacities of algae and protozoa

In spite of algae and protozoa are the important members of the microbial community in both aquatic and terrestrial ecosystems, reports are scanty regarding their involvement in hydrocarbon biodegradation. Walker et al. isolated an alga, Prototheca zopfi which was capable of utilizing crude oil and a mixed hydrocarbon substrate and exhibited extensive degradation of n-alkanes and isoalkanes as well as aromatic hydrocarbons. Cerniglia and Gibson observed that nine cyanobacteria, five green algae, one red alga, one brown alga, and two diatoms could oxidize naphthalene. Protozoa, by contrast, had not been shown to utilize hydrocarbons, however, protozoa population have been shown to significantly reduce the number of bacteria available for hydrocarbon removal so their presence in a biodegradation system may not always be beneficial . Rogerson and Berger found no direct utilization of crude oil by protozoa cultured on hydrocarbon-utilizing yeasts and bacteria. Overall, the limited available evidence does not appear to suggest an ecologically significant role for algae and protozoa in the degradation of hydrocarbons in the environment. Some research has demonstrated that certain fresh algae (e.g. Chlorella vulgaris, Scenedesmus platydiscus, S. quadricauda and S. capricornutum) are capable of uptaking and degrading PAHs.

Information on the interactions between pesticides andalgae were compiled by Kobayashi and Rittman, showing that not only algae were capable of bioaccumulating pesticides, but they were also capable of biotransforming some of these environmental pollutants.

Degradation of azo dyes by C. vulgaris and C. pyrenoidosa was made using dyes as carbon and nitrogen sources, but this was dependent on the chemical structure of the dyes. The degradation was found to be an inducible catabolic process. C. vulgaris, Lyngbyala gerlerimi, Nostoc lincki, Oscillatoria rubescens, Elkatothrix viridis and Volvox aureus were able to decolorize and remove methyl red, orange II, G-Red (FN-3G), basic cationic, and basic fuchsin.

Chemical decolorization of Azo Dyes by peroxide	Azo dyes

Azo dyes and pigments are used to color most textiles and leathers. They are dangerous to work with, giving off carcinogenic amines. The name Azo is derived from the Greek a (not) + zoe (to live). With a name like that, it’s no wonder these dyes have an adverse affect on water resources, soil fertility, and eco-system integrity. The industry also uses significant amounts of bleaches, acids, alkalis, salts, stabilizers, surfactants, fire retardants, softeners, starches, heavy metals, and an assortment of dyes (acid, basic, disperse, mordant, reactive, sulphur dye, pigment, and vat). Most of these chemicals are applied using water as a medium. With the price of water consumption and effluent disposal increasing, some companies are beginning to look at ways to reduce water usage and find viable ways to treat effluent, while many dye houses will continue to use up local water supplies and dump untreated toxic wastewater into streams and rivers until the cows come floating home.

Species of Chlorella, Anabaena inacqualis, Westiellopsis prolifica, Stigeoclonium lenue, Synechoccus sp. tolerate heavy metals and several species of Chlorella, Anabaena and marine algae have been used for the removal of heavy metals, but the operational conditions limit the practical application of these organisms. Metals are taken up by algae through adsorption. Metal chelation by unicellular algae has been reported. Biosorption of heavy metals by brown algae is also known since a long time, this includes sorption of heavy metals by a number of cell wall constituents such as alginate and fucoidan. Most of the research in this area has been carried out on marine and soil algae. The microalga S. incrassatulus was reported to remove Cr(VI), Cd(II) and Cu(II) in continuous cultures. Green algae were also reported in heavy metals bioremediation, C. sorokiniana for Cr(III) removal.

The protozoa are the main grazer on the degrading bacteria for organic contaminants, so the interaction between protozoa and degrading bacteria will affect the result of bacteria degradation directly. Mattison and Harayama constructed a model for the food chain in order to study the influence of grazing bacteria of protozoa flagellate Heteromita globosa on the biodegradation of benzene and methylbenzene. They found that during the logarithmic growing period of flagellate population, the degrading rate of benzene and methylbenzene has improved 8.5 times by bacteria than before. The protozoa infusorians can obviously accelerate the biodegradation of heterogenous substances in the environment such as PAH. For example, the degradation rate of naphthalene can be improved by 4 times than before. There are several possible hypotheses about the mechanism of protozoa accelerating bio-degradation of organic contaminants, which mainly include the following six parts:

• the nutrient mineralization which improves the turnover of nutrients;
• bacteria activation which controls the quantity, grazes the aged cells or excretes active substance;
• selective grazing which reduces the competition to the resource and space and thus is good for the growth of degrading bacteria;
• physical disturbance which can increase oxygen content and the surface of degraded matters;
• direct degradation which can excrete special enzymes participating in the degradation;
• sym-metabolism which offers energy and carbon resource for the bacteria during the degradation.