WO2013149662A1

WO2013149662A1 - Inoculated bioplastic-based moving bed biofilm carriers

Info

Publication number: WO2013149662A1
Application number: PCT/EP2012/056218
Authority: WO
Inventors: Cesare ACCINELLI; Mariangela MENCARELLI; Maria Ludovica SACCA'
Original assignee: Hera S.P.A.
Priority date: 2012-04-04
Filing date: 2012-04-04
Publication date: 2013-10-10

Abstract

The present invention relates to a moving bed biofilm carrier (MBBC) having the shape of a Kaldnes-like carrier, characterized in that it is inoculated with one or more microbial strain(s) and is manufactured from bioplastic. In addition, the present invention is also directed to the use of the bioplastic-based moving bed biofilm carriers according to the invention for the bioremediation of wastewater or water contaminated by pollutants such as for example hydrocarbons and oil-derivatives, pharmaceuticals, pesticides, phytosanitary compounds, herbicides, pesticides and dyes.

Description

INOCULATED BIOPLASTIC-BASED MOVING BED BIOFILM CARRIERS

Increasing concerns over preserving water quality have stimulated the development of a variety of technologies for reducing the environmental impact of human activities on this non-renewable and vulnerable resource. Among the different approaches that have been proposed in these last decades, the technology known as moving bed biofilm reactors (MBBR) has been successfully applied in the clean-up of wastewaters from urban, industrial and agricultural uses. MBBR is based on the usage of small cylindrical carriers (MBBC, moving bed biofilm carriers), which move freely in the wastewater reactor. These cylinder- shaped carriers are specifically designed to provide a large surface area for the growth of attached microbial biofilm. Typically, a variable fraction of the total reactor volume is filled with these MBBC which are then kept in continuous movement by aeration or mechanical stirring of the fluid. First proposed in the early 1990s by Norwegian scientists mainly for the purpose of BOD/COD abatement of domestic wastewater in small wastewater treatment plants, this technology is now applied worldwide in a variety of different situations. In fact, other than for the reduction of BOD/COD and total nitrogen from wastewater, the use of MBBR technology has been successfully extended in aquaculture and for the removal of organic xenobiotics (i.e. pharmaceuticals, pesticides, dyes, etc.) from contaminated waters.

In the MBBR, microbial growth is mainly confined to the surface of MBBC and especially to their sheltered internal surfaces. This results in the formation of a stable and sheltered biofilm, protecting microbes from alteration of wastewater parameters which are caused by variability of effluent characteristics and operating procedures. Other than promoting high microbial activity, major advantages of MBBR also include reduced head losses and elimination of backwashing costs. In their original design, MBBC consist of small cylinders (typically having a 10-60 mm diameter and a 3-50 mm height) generally having longitudinal fins on their external surface. A series of flat surfaces or rods protruding from the internal surface of the cylinder toward the central axis thereof (either contacting with each other or not) are usually present, to increase the surface available for the growth of the microbial biofilm. Further cylindrical surfaces, concentric to each other and to the most external cylindrical surface can be present in the internal cavity of the cylinder. When more than one cylindrical surfaces are present, they are linked to each other through flat surfaces or rods. Such MBBC are known as "Kaldnes-like cylinders" or "Kaldnes-like carriers". Although many variations from the original design have been proposed, most of the MBBC launched in the market during these last two decades have maintained a cylinder-like shape.

The MBBC actually available in the market or however described in the scientific literature, including the Kaldnes-like carriers, are made of petroleum-based plastic (i.e. high-density polyethylene and other polymers). In particular, they are generally made of high-density polyethylene, which has a density slightly less than water, thus facilitating the movements of the carriers inside the fluid mass.

On the other hand, the fact that the MBBC are made of plastic is associated to some unpleasant drawbacks. As a first issue, the manufacture of plastic MBBC depends on petroleum-based raw materials, i.e. a nonrenewable source. In addition, after the end of their life-cycle, these products are not easily manageable, first of all for the very fact that they are plastic-based, non biodegradable products, but also because after their use they can be contaminated. Another drawback, which arises as a consequence of those mentioned above, is that the disposal of these carriers is expected to be costly and technically not easily feasible, although no indications concerning this aspects have been reported in the technical and scientific literature. Advancements in polymer science have resulted in the discover and/or synthesis of affordable and feasible polymers from natural sources which are used for producing biodegradable and sustainable plastic (bioplastic). Although approximately 140 million tons of petroleum-based polymers such as polyethylene, polypropylene, polystyrene, poly(ethylene terephthalate), polycarbonate and poly(vinyl chloride) are annually used in a variety of industrial and day-to-day plastic applications, bioplastic industry is increasing its importance in a variety of practical applications. Among the different bioplastic materials that have been launched in the market, polylactic acid (PL A, also known as polylactide) and starch-based bioplastics occupy a relevant importance in the replacement of petrol-based plastic products. Both PLA and the starch-based bioplastics are produced from renewable agricultural resources, mainly starch-rich crops (i.e. corn).

In more recent years, the concept of having an attached biofilm on moving bed carriers has been extended to other practical and technical solutions. For instance, it has been proposed to replace Kaldnes-like cylinders with less costly granules. In a restricted number of cases, these granules can be made of bioplastic (Accinelli et al., 2010; Chu and Wang, 2011).

In the context of the present invention, the expressions "Kaldnes-like cylinder", "Kaldnes-like carrier", "carrier having/with a Kaldnes-like shape/structure" "carrier having/with the shape/structure of Kaldnes-like carrier" are meant to refer to a carrier having exclusively the shape of a Kaldnes-like carrier as described above which identifies in general a carrier having a cylindrical shape wherein one or more flat surfaces or rods are present, protruding internally, externally, or both internally and externally with respect to the cylindrical surface.

Kaldnes-like carriers are described in detail for example in WO 91/11396 (also published as EP 0575314) and WO 95/25072 (also published as EP 0750591) herein inclosed by reference. The main purpose of the present invention is to provide an alternative moving bed biofilm carrier (MBBC) with a Kaldnes-like shape which is not made of traditional plastic of petroleum origin. In particular, an aim of this invention is to provide a MBBC with a Kaldnes-like shape which can be disposed of, after the end of its life-cycle, in an easy manner and without incurring in high costs. Another aim of the invention is to provide a MBBC with a Kaldnes-like shape which, at the end of its life-cycle, is not contaminated by pollutants agents. A further purpose of the present invention is to provide a carrier with a Kaldnes-like shape which can be employed in the processes of wastewater treatment and bioremediation of contaminated waters. Within this purpose, the present invention aims at providing a carrier with a Kaldnes-like shape which does not undergo chemical degradation in the presence of chemical pollutants in the contaminated waters to be treated.

The above purposes, as well as other purposes which will appear in the following, are achieved by a moving bed biofilm carrier having the shape of a Kaldnes-like carrier, characterized in that it is inoculated with one or more microbial strain(s) and is manufactured from bioplastic.

In the context of the present invention, the term "bioplastic" is meant to indicate a plastic derived from renewable biomass sources, such as vegetable fats and oils or starch, rather than from petroleum and which is biodegradable, i.e. degrades in natural environments.

Description of the figures:

Figure 1 represents an exemplary structure of a Kaldnes-like moving bed biofilm carrier.

Figure 2 shows the effect of non inoculated and inoculated bioplastic- based MBBC according to the invention on ¹⁴C0₂ evolution from wastewater samples spiked with bisphenol A (BPA). Bars represent standard deviations of the means.

Figure 3 shows the effect of non inoculated and inoculated bioplastic- based MBBC according to the invention on ¹⁴C0₂ evolution from wastewater samples spiked with oseltamivir carboxylate (OC). Bars represent standard deviations of the means.

Figure 4 shows the effect of non inoculated and inoculated bioplastic- based MBBC according to the invention on ¹⁴C0₂ evolution from wastewater samples spiked with atrazine (ATZ). Bars represent standard deviations of the means.

Detailed description of the invention:

In a first aspect, the present invention relates to a moving bed biofilm carrier (MBBC), having the shape of a Kaldnes-like carrier, characterized in that it is inoculated with one or more microbial strain(s) and is manufactured from bioplastic.

The bioplastic-based MBBC of the invention can be used in moving bed biofilm reactors (MBBR) for bioremediation processes of wastewater or contaminated water.

In a preferred embodiment of the invention, the bioplastic-based carriers of the invention having the shape of a Kaldnes-like carriers are cylinders having an internal diameter of 2 cm, a thickness of 0.25 cm and an external diameter of 2.25 cm. The external surface can preferably be provided by prism-shaped fins to increase the surface of the carrier wherein the microbial biofilm adheres. In a preferred embodiment, the prism-shaped fins placed on the external surface of the cylinder can have a base of 0.25 cm and a height of 0.3 cm.

A certain number of rods can be present in the internal cavity of the cylinder; preferably, four radially arranged rods can be present. In a further preferred embodiment, these rods can be hollow. In addition, also these rods can be provided with prism-shaped fins, to further increase the surface available for microbial growth in the internal cavity of the cylinder, which is the most suitable for microbial growth, being sheltered from the external environment. Preferably, the prism-shaped fins placed on the rods in the internal cavity of the cylinder can have a base of 0.05 cm and a height of 0.055 cm. All the linear dimensions recited above can be varied up to 20% of the above values (either by increasing or by decreasing them), maintaining the proportion of the above lengths to one another. Due to the above recited dimensions, the resulting volume of the cylinders amounts to 2.34 cm³ (±20%).

The bioplastic-based carriers of the invention are made of the biodegradable plastic polylactide (PL A) and/or one or more starch-based bioplastic(s). As an example, the starch-derived bioplastic Mater-Bi® (manufactured by Novamont S.p.A, No vara, Italy) can be used. In an embodiment, the carriers are entirely made of polylactide; in another embodiment, the carriers are entirely made of one or more starch-based bioplastic(s). In a further embodiment, the cylindrical body of the carrier is made of polylactide and the internal rods are made of one or more starch- based bioplastic(s). Alternatively, in another embodiment, the cylindrical body of the carrier is made of one or more starch-based bioplastic(s) and the internal rods are made of polylactide. The bioplastic-based carriers can further be made, either totally or partially, of other biodegradable polymers, such as polyhydroxyalkanoates (PHAs) and poly(3-hydroxybutyrate-co-3- hydroxy valerate (PHBV).

Depending on the particular composition of the bioplastic-based carriers, the density of the material which they are made of can vary between 0.03 and 0.3 g/cm³.

The bioplastic-based carriers of the invention can be manufactured by common extrusion processes. As stated above, the carriers of the invention are inoculated with one or more selected microbial strain(s). The microbial inoculum is put into and/or onto the rods protruding in the internal cavity of the cylinders. After inoculation, the cylinders are sealed on both sides with plugs made of one or more starch-based bioplastic(s) . Sealing the sides of the cylinders serves the purpose of protecting the inoculated microorganisms to allow a gradual adaptation of the microorganisms themselves to the wastewater or contaminated water and an initial increase of the biomass.

The density of the one or more bioplastic(s) which the plugs are made of affects the time needed for such plugs to disintegrate, thus allowing the inoculated microorganisms to contact the wastewater or contaminated water. The higher the density of the plugs is, the slower is their disintegration.

Generally speaking, plugs having low density (e.g. 0.03 g/cm³) and a spongy consistency are desired, which degrade in about three days, thus rapidly contacting the microorganisms to the wastewater or contaminated water. To obtain low density, spongy bioplastic plugs, the final extrusion step of the manufacturing process thereof is performed at high speed. On the other hand, if a longer time and a more gradual approach is needed for the microorganisms to adapt to the wastewater or contaminated water, plugs having a higher density can be advantageously used (e.g. 0.3 g/cm³, which require up to six days for their degradation to occur).

As already stated above, the main role of the carriers in a MBB is to provide a support for the growth of microorganism colonies on their surface, resulting in the formation of a stable and sheltered biofilm.

However, Applicant surprisingly found that carriers having a

Kaldnes-like shape and manufactured with biodegradable materials allow to obtain a particularly improved effect in terms of working efficacy of these carriers. In fact, on one hand the Kaldnes-like shape offers an optimal solution in terms of the ratio between volume and surface available for the growth of the microorganisms on the carrier itself, thus maximizing the amount of the biomass and optimizing the growth conditions of the microorganisms. On the other hand, in view of the biodegradable material which they are made of, the carriers of the invention also represent a carbon source, thus providing a nutritional substrate for the growing microorganisms, contrary to the traditional, oil-derived plastic carriers, which represent a mere physical support for the growth of microorganisms.

The combination of the (structurally) ideal Kaldnes-like shape with the biodegradable, organic material which the carriers of the invention are made of results in a synergistic effect, whereby an active and stable biofilm is formed.

The growth of the biomass on the bioplastic-based carriers of the invention is greatest on the rods protruding towards the internal cavity of the cylinders and on the internal surface(s) of the cylinders. The rods and the internal surface(s) of the cylinders are in fact less exposed to stress conditions such as abrasion due to the water flux or to collisions with other carriers and predation by microorganisms like protozoa or metazoa which can be present in the wastewater or contaminated water. As a consequence of the stress conditions mentioned above, however, the microbial biofilm may undergo a partial detachment from the surface of the carriers, which results in the release of portions of the biofilm which remain suspended into the wastewater or contaminated water inside the reactor.

In another aspect, the present invention relates to the use of the bioplastic-based moving bed biofilm carriers of the invention for the bioremediation (i.e. the removal of pollutants performed by microorganisms) of wastewater or contaminated water. Preferably, the wastewater can be selected from the group consisting of biological, chemical and biological/chemical wastewater. For example, such wastewater can be wastewater from the urban, industrial and agricultural environments.

In a preferred embodiment, the bioplastic-based MBBC of the invention can be used for the abatement of BOD/COD from wastewater. In another preferred embodiment, the bioplastic-based MBBC of the invention can be used for the reduction of carbon and/or nitrogen and/or phosphorous content from wastewater.

The bioplastic-based MBBC of the invention are also used for the bioremediation of waters contaminated by pollutant agents. In a preferred embodiment, such pollutant agents can be selected from the group consisting of hydrocarbons and oil-derivatives. In another preferred embodiment, such pollutant agents can be synthetic compounds. In a more preferred embodiment, such synthetic compounds are selected from the group consisting of xenobiotics, pharmaceuticals, pesticides, phytosanitary compounds, herbicides and dyes.

Bioremediation is based on a natural process, which exploits the ability of some microorganisms to degrade chemical substances representing environmental contaminants or pollutants. Contaminants or pollutants of organic origin can be either completely degraded by microorganisms to carbon anhydride and water (mineralization) or converted into compounds with a lower toxicity. Bioremediation is usually employed to decontaminate contaminated environments such as soil, superficial water, groundwater and the treatment equipments themselves.

The microorganisms used in bioremediation are able to exploit chemical compounds representing a pollutant or a contaminant as a source of energy for their growth. Many microorganisms are known for possessing a catabolic activity towards substances considered toxic or environmental contaminants.

Bisphenol A is widely used in the synthesis of plastics and plastic additives. Concerns have been raised about the presence of Bisphenol A in consumer products, as it interferes with the endocrine system exerting weak but detectable hormone-like properties, which become particularly dangerous when fetuses, infants and young children are exposed to this product. Bisphenol A is also involved in a decreased fertility of adult men. Many bacterial species have been isolated and characterized as capable of degrading Bisphenol A. Some of these bacteria belong to the genuses Bacillus spp. and Pseudomonas spp.; another example of Bisphenol A degrading microorganism is Sphingomonas bisphenolicum. In addition, also ammonia-oxidating bacteria, such as Nitrosomonas Europaea have the capacity to degrade Bisphenol A, as well as the active pharmaceutical ingredient ibuprofen.

17P-estradiol is also a toxic compound which interferes with the endocrine system and which is not degraded in conventional water treatment systems. Bacterial species belonging to the genuses Aminobacter spp., Brevumdimonas spp., Escherichia spp., Flavobacterium spp., Microbacterium spp., Nocardioides spp., Rhodococcus spp. and Sphingomonas spp. have been isolated and characterized as capable of degrading 17P-estradiol.

A species of the Arthrobacter genus has been used to degrade the herbicide atrazine in a contaminated soil. This Arthrobacter species is likely to be effectively used in other contaminated areas, for example in the equipment for wastewater treatment. In addition, some bacterial species, such as Pseudomonas spp. and Rhodococcus spp. are known to be capable of degrading herbicides of the s-triazine family.

Bioremediation is largely applied to environmental areas contaminated by hydrocarbons and oil-derivatives in general through hydrocarbon-degrading bacteria. Among these ones, gamma-Proteobacteria and Cytophaga-Flavobacteria are capable of degrading phenol and derivatives thereof.

Polycyclic aromatic hydrocarbons (PAH) such as naphthalene and phenanthrene are environmental contaminants highly dangerous for the human health because of their potential mutagen activity. Bacterial species such as Alcaligenes denitrificans, Mycobacterium spp., Pseudomonas putida and Streptomyces spp. have proved capable of degrading naphthalene. Phenanthrene can instead be metabolized by bacterial species such as Aeromonas spp., Alcaligenes faecalis, Arthrobacter polychromogenes and Micrococcus spp.

Further to bacteria, also several fungal species have a catabolic action towards chemical contaminants. The lignino-lythic basidiomycete Phanerochaete chrysosporium is one of the most well-studied degrading fungi, in view of its degrading properties against a wide number of chemical contaminants, in particular of antibiotics.

In the present invention, the one or more microorganism(s) to be inoculated on the bioplastic-based moving bed biofilm carrier according to the invention is selected among the species of microorganisms known for their decontaminating activity. The selection of the one or more microorganism species to be inoculated in the carrier is based on the kind of contaminant(s) present in the wastewaters or contaminated waters to be treated. In particular, the one or more microorganism(s) can be bacteria and/or fungi. In a preferred embodiment, the one or more microorganism(s) are ammonia-oxidating bacteria. In another preferred embodiment, the one or more microorganism(s) are hydrocarbon-degrading bacteria. More preferably, the one or more microorganism(s) are poly cyclic aromatic hydrocarbon-degrading bacteria. Naphthalene-degrading bacteria and phenanthrene-degrading bacteria are even more preferred.

More particularly, among fungi, the microorganism can preferably be Phanerochaete chrysosporium . Among bacteria, the microorganism(s) can preferably be selected from the group consisting of Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Aminobacter spp., Arthrobacter spp., Bacillus spp., Brevumdimonas spp., Cytophaga-Flavobacteria spp., Escherichia spp., Flavobacterium spp., Mycobacterium spp., Microbacterium spp., Micrococcus spp., Nocardioides spp., Pseudomonas spp., gamma-Proteobacteria spp., Rhodococcus spp., Sphingomonas spp., Streptomyces spp.

In a preferred embodiment, (one of) the microorganism(s) can be Sphingomonas bisphenolicum . In another preferred embodiment, (one of) the microorganism(s) can be Nitrosomonas Europaea. In another preferred embodiment, (one of) the microorganism(s) can be Pseudomonas putida. In a further preferred embodiment, (one of) the microorganism(s) can be Arthrobacter polychromogenes . In still another preferred embodiment, (one of) the microorganism(s) can be Alcaligenes denitrificans . In still another preferred embodiment, (one of) the microorganism(s) can be Alcaligenes faecalis.

The Agrobacterium sp. BPA strain can also be (one of) the preferred microorganism(s) inoculated on the carriers of the present invention.

The Flavobacterium sp. OC strain can also be (one of) the preferred microorganism(s) inoculated on the carriers of the present invention.

The Pseudomonas sp. ADP strain can also be (one of) the preferred microorganism(s) inoculated on the carriers of the present invention.

As already said above, the one or more microbial strain(s) with which the bioplastic-based MBBC are inoculated are chosen on the basis of the specific contaminant(s), xenobiotic(s) and/or compound(s) contained in the wastewater or contaminated water.

EXAMPLE 1 : Preparation of bioplastic moving bed carriers

Moving bed biofilm carriers (MBBC) manufactured with Ingeo™ polylactide (PLA) biopolymer (Nature Works LLC, Minnetonka, MN, USA) and consisting of cylinders with internal diameter and high of 2 cm were prepared for this study. A number of four intersecting hollow rods (0.5 cm internal diameter) were placed into the internal volume of the cylinder. Rods were produced with the starch- derived bioplastic Mater-Bi® (Novamont S.p.A, Novara, Italy). Rods were cut to a length of 2.2 cm and then inserted in the cylinder which was perforated for keeping the sticks in a fixed position. Prepared MBBC (Figure 1) were then autoclaved at 120 °C for 15 min. In the case of inoculated MBBC, a disk of freeze-dried cells was inserted in the middle of the stick. The edges of this inoculated sticks were capped with autoclaved plugs. Plugs were obtained from spongy Mater- Bi®. All the operations were conducted under sterile conditions.

EXAMPLE 2: Microbial inoculation of bioplastic carriers Bioplastic carriers were inoculated with the following bioremediation bacteria strains: Agrobacterium sp. BPA (capable to degrade bisphenol A, i.e. BPA), Flavobacterium sp. OC (capable to degrade oseltamivir carboxylate, i.e. OC), and Pseudomonas sp. ADP (capable to degrade atrazine, i.e. ATZ). The two latter strains were isolated in other studies (Mandelbaum et al., 1993; Accinelli et al., 2010) and are capable to mineralize OC and ATZ, respectively. The former strain was isolated from soil using a conventional enrichment procedure described in Accinelli et al., 2010 and selected for its capability to mineralize BPA.

After growing overnight in Luria-Bertani (LB) broth, cells of the selected bacterial strains were harvested by centrifugation at 10,000 g for 10 min and washed twice with sterile phosphate buffer saline (PBS). Cells were freeze-dried for 20 h at -50°C and then size of the inoculum (number of viable cells per mass of freeze-dried culture) was determined by spread- plate on LB agar. A mass of inocula corresponding to a total of approximately 10¹² colony forming units was then introduced into autoclaved sticks.

EXAMPLE 3: Removal of bisphenol A from wastewater using inoculated and bioplastic-based moving bed biofilm carrier

Bioplastic-based MBBC inoculated with the bacterial strain

Agrobacterium sp. BPA were used for the removal of bisphenol A (BPA) from wastewater samples collected from the outflow of the main biological tank of municipal wastewater treatment plant (WWTP) of Bologna, Italy.

Sampling operations were conducted in September 2011 using aseptic techniques. Samples were stored at 4°C for no longer than 2 days. Prior to use, all samples were left at 20°C overnight. Detailed characteristics of this wastewater are summarized in Accinelli et al. 2010.

Mineralization of BPA was monitored in 2.5-L hermetically sealed jars containing 400 mL of wastewater. Each jar was equipped with a suspended glass vials containing 4 mL of a 1 M NaOH solution to trap ¹⁴C0₂. Wastewater samples were spiked with a solution of unlabeled (chemical purity > 99%; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and ring-¹⁴C(U) radiolabeled BPA (32.4 MBq*mg^_1; radiopurity 99%; American Radiolabeled Chemicals Inc., St. Louis, MO) to give a final concentration of 10 μg*mL^"1. A number of eight MBBC were introduced to each flask and incubated for 10 days. The experiment was performed with non- and inoculated MBBC. Samples without MBBC were included as control. All the samples were kept in continuous movement by mechanical stirring. Vials containing the NaOH solution were replaced every day thus aerating the samples. Trapped ¹⁴C0₂ was determined by mixing a 1-mL aliquot of NaOH solution with 4 mL of Hi Safe 3 scintillation cocktail (Perkin-Elmer, Boston, MA, USA), and the amount of radioactivity was measured by liquid scintillation counting using a TriCarb400 liquid scintillation counter (Perkin-Elmer, Meriden, CT). Prior to analysis, samples were kept in the dark for 12 hours.

Mineralization of BPA expressed as ¹⁴C0₂ evolution is shown in Figure 2. At the end of the 10-day incubation period, approximately 17% of the initial radioactivity was recovered as ¹⁴C0₂ from wastewater samples not containing MBBC (control). Cumulative ¹⁴C0₂ evolution from samples containing non- and inoculated carriers accounted for approximately 23% and 27% of the initial radioactivity, respectively. Although a variety of techniques for the removal of BPA have been explored in the last decades, none has been focused on the application of the moving bed biofilm reactor technology.

EXAMPLE 4: Removal of the antiviral drug oseltamivir from wastewater using inoculated and bioplastic-based moving bed biofilm carrier

The same experimental scheme and procedures described in EXAMPLE 3 were adopted to assess the efficiency of inoculated bioplastic- based MBBC for the removal of oseltamivir carboxylate (OC), the active form of the antiviral drug oseltamivir phosphate. Wastewater samples were spiked with a solution of unlabeled (chemical purity > 98%) and ^Relabelled OC (radiopurity > 98%, specific activity 4.96 MBq*mg^"1) to give a final concentration of 10 μg*mL^"1. Both chemicals were provided by F. Hoffmann-La Roche Ltd (Basel, Switzerland). Bioplastic-based MBBC were inoculated with the OC-degrading Flavobacterium sp.

Mineralization of the antiviral drug OC proceeded slower than that of BPA (Figure 3). In control samples, at the end of the 10-day incubation period, approximately 7% of the initial radioactivity was recovered as ¹⁴C0₂ from control. Application of non-inoculated MBBC resulted in an 1.4-fold increase of the cumulative ¹⁴C0₂ evolution. Mineralization of OC was further stimulated with inoculated MBBC. More precisely, inoculating the bioplastic carriers with the bacterium Flavobacterium sp. which was previously selected for its bioremediation capability, led to an approximately 25% increase of cumulative ¹⁴C0₂ with respect to the non- inoculated carriers. These results are consistent with the current knowledge on the fate of OC in aquatic environments. Similarly to a large variety of other organic xenobiotics, mineralization of OC proceeds slowly in water. However, when the size of the microbial community is artificially increased (i.e. by adding sediments), mineralization is also stimulated (Accinelli et al., 2007; Sacca et al, 2009).

EXAMPLE 5: Removal of the pesticide atrazine from wastewater using inoculated and bioplastic-based moving bed biofilm carrier

Inoculated bioplastic-based MBBC were also evaluated for their efficiency to remove the pesticide atrazine (ATZ) from wastewater. Using the same protocol described in EXAMPLES 3 and 4, a mixture of unlabeled (chemical purity > 99%; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and ring-14C(U) radiolabeled ATZ (1.72 MBq*mg^_1; radiopurity 99%; American Radiolabeled Chemicals Inc., St. Louis, MO) was added to wastewater samples to provide a final concentration of 10 μg*mL^"1. Evolution of ¹⁴C0₂ was monitored as described above. Within the 10-day period, ATZ mineralization accounted for approximately 3.5% of the initial added radioactivity (Figure 4). As observed with BPA and OC, application of both non- and inoculated bioplastic MBBR led to a significant increase of atrazine mineralization. More specifically, in relative terms, inoculated MBBC showed higher efficiency with ATZ than with the other two combination of xenobiotics/bioremediation strains. As shown in Figure 4, cumulative ¹⁴C0₂ evolution from samples containing non inoculated carriers accounted for 6.3% of the total initial radioactivity. When carriers were inoculated with the ATZ-degrading strain Pseudomonas sp. ADP, cumulative ¹⁴C0₂ evolution accounted for 8.7% of the initial added radioactivity.

EXAMPLE 6: formation of attached microorganisms

At the end of the 10-day incubation period, MBBC were removed from cylinders and washed twice in sterile phosphate buffer. After drying at 35°C, MBBC were transferred to 50-mL centrifuge tubes containing 30 mL of PBS and of glass beads. Tubes were vortexed for 5 min and then horizontally shaken at 250 rpm for 2 hours. Tubes were then centrifuged at 10,000 x g for 5 min. The procedure was repeated replacing the shacking step with sonication of the samples at 30 kHz for 10 min. Obtained pellets were bulked together, dried at 40 °C overnight and then used for quantitative PCR (qPCR) analysis. DNA isolation was achieved using the commercial kit UltraClean Soil DNA (MoBio Laboratories Inc., Solana Beach, CA). Quantitative PCR (qPCR) was performed following the procedure described in Sacca et al. (2009). Briefly, PCR amplification was performed using the primer pair Eub338/Eub518, targeting a fragment of the conservative bacterial 16S rRNA gene. The sequences of the primers are:

Eub338: ACT CCT ACG GGA GGC AGC AG (SEQ. ID. No: 1); Eub518: ATT ACC GCG GCT GCT GG (SEQ. ID. No: 2).

Such primers are reported in Frierer et al. (2005). Each 25 μΕ qPCR reaction contained 2 μΕ of DNA, 12.5 μΕ of 2* TaqMan Universal PCR Master Mix (Applied Biosy stems, CA), and 0.2 μΜ of each primer. Thermocy cling conditions were as follows: 2 min at 50°C, 10 min at 95°C, cycles of 15 s at 95°C and 1 min at 60°C. Reactions were performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Gene copies were estimated by comparison of cycle threshold values obtained from known amounts of DNA. The same procedure was used for enumerating the copy numbers of atzC gene from inoculated MBBC of the ATZ experiment. The sequences of primers targeting the atzC gene were the following:

AtzCqF: TCG TAG CCT TTG CAC AGA GTG GAT T (SEQ. ID.

No: 3);

AtzCqR: TTT TCC CGC GTA GCA GGA TCA AC (SEQ. ID. No:

4)·

Results of the qPCR analysis targeting a fragment of the conservative 16S rRNA bacterial gene are summarized in Table 1 below. Similarly to other studies, quantification of the PCR-amplified 16SrRNA products is here assumed to give an estimation of the size of the bacterial community. The average copy number of the amplified 16S rRNA fragments recovered from control (no MBBC) wastewater samples was of loglO copies per ng of DNA, with no significant differences among the three chemicals. Detrimental effects of these chemicals on wastewater bacteria are consequently excluded. Considering the qPCR data in terms of copy numbers per unit of volume of liquid (wastewater) or solid (MBBC) phase, it appears clear that the moving biodegradable carriers promoted the growth of bacteria and, most importantly, the formation of bacterial biofilms on their surface (Figure 2). This stimulatory effect was more pronounced in samples containing inoculated MBBC, regardless of chemicals and bacterial strains. QPCR data indicated the formation of adhering bacterial biofilm on MBBC, with inoculated carrier having a superior capability. Using qPCR, the copy number of the gene atzC which is harbored by Pseudomonas sp. ADP was also quantified. Higher abundance of this gene was observed in DNA samples recovered from MBBC (copy number of the AtzC gene: 1.2 x 10⁹) than from the liquid phase (copy number of the AtzC gene: 1.4 x 10⁵) , thus demonstrating the reliability of these carriers for promoting the adherence of newly introduced bacterial strains on their surface.

Non-inoculated Inoculated

MBBC MBBC

Control 87 ± 7.7 182 ± 6.1

BPA 71 ± 11.3 201 ± 14.4

OC 97 ± 9.5 239 ± 11.1

ATZ 80 ± 8.8 240 ± 9.9

201 ± 12.9

(31.2 ± 6.1)

Table 1. Total bacterial DNA (nanograms) from attached biofilm of bioplastic-based moving biofilm bed carriers (MBBC) recovered from wastewater samples spiked with bisphenol A (BPA), oseltamivir carboxylate (OC) or atrazine (ATZ) and incubated for 10 days. The total amount of DNA fragments corresponding to the atzC gene are reported in parenthesis. Number is means of three replicates ± STD.

In the light of the above, it has been ascertained that the bioplastic- based carriers with Kaldnes-like shape provided by the present invention are capable of solving the established purposes in an effective manner. In fact, the carriers according to the invention, being made of bioplastic, do not exploit a non-renewable source and, after the end of their life-cycle can be disposed of in an easier and more economical manner with respect to disposal of carriers made of oil-derived plastic. In addition, at the end of their life-cycle, the carriers of the invention are not contaminated by pollutants agents as the one or more microorganism(s) inoculated thereon are capable of degrading the pollutant substances to be removed. In addition, it has been demonstrated that the carriers of the invention can be employed in the bioremediation of wastewater and contaminated water, effectively improving the degradation of the pollutants performed by the microorganism(s) inoculated thereon. The bioplastic-based carriers of the invention in fact have been shown to provide a suitable and sheltered support wherein the microbial biomass can grow on a small volume thus improving the efficacy of the bioremediation of wastewaster or contaminated water.

Claims

1. A moving bed biofilm carrier (MBBC), having the shape of a Kaldnes-like carrier, characterized in that it is inoculated with one or more microbial strain(s) and is manufactured from bioplastic.

2. The carrier of claim 1 , wherein the one or more bioplastic(s) is selected from the group consisting of a polylactide (PLA), a starch-based bioplastic, a polyhydroxyalkanoate (PHAs), a poly(3-hydroxybutyrate-co-3- hydroxyvalerate (PHBV) and combinations thereof.

3. The carrier of any of claims 1 or 2, wherein the one or more microbial strain(s) is selected from microorganisms capable of degrading one or more chemical pollutant(s).

4. The carrier of claim 3, wherein the one or more microbial strain(s) is selected from the group consisting of a hydrocarbons and oil-derivatives- degrading microbial strain, a xenobiotic- degrading microbial strain, a pharmaceutical-degrading microbial strain, a pesticide-degrading microbial strain, a phytosanitary compound-degrading microbial strain, a herbicide- degrading microbial strain and a dye-degrading microbial strain.

5. The carrier of any of claims 3 or 4, wherein the one or more microbial strain(s) is selected from the group consisting of Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Aminobacter spp., Arthrobacter spp., Bacillus spp., Brevumdimonas spp., Cytophaga-Flavobacteria spp., Escherichia spp., Flavobacterium spp., Mycobacterium spp., Microbacterium spp., Micrococcus spp., Nocardioides spp., Pseudomonas spp., gamma-Proteobacteria spp., Rhodococcus spp., Sphingomonas spp., Streptomyces spp., Nitrosomonas Europaea, Phanerochaete chrysosporium and combinations thereof.

6. The carrier of any of claims 3 to 5, wherein the one or more microbial strain(s) is selected from the group consisting of Agrobacterium sp. BPA, Flavobacterium sp. OC, Pseudomonas sp. ATZ and combinations thereof.

7. Use of the carrier of any of the previous claims for the bioremediation of wastewater or contaminated water.

8. The use according to claim 7, wherein said wastewater or contaminated water is selected from the group consisting of biological, chemical and biological/chemical wastewater or contaminated water.

9. The use according to any of claims 7 or 8, wherein said wastewater or contaminated water is selected from the group consisting of wastewater or contaminated water from the urban environment, wastewater or contaminated water from the industrial environment and wastewater or contaminated water from the agricultural environment.

10. The use according to one or more of claims 7 to 9, wherein said treatment of the wastewater or contaminated water comprises the abatement of BOD/COD.

11. The use according to one or more of claims 7 to 9, wherein said treatment of the wastewater or contaminated water comprises the reduction of carbon and/or nitrogen and/or phosphorous content.

12. The use according to one or more of claims 7 to 9, wherein said treatment of the wastewater or contaminated water comprises the degradation of one or more pollutant agent(s).

13. The use according to claim 12, wherein said one or more pollutant agent(s) is selected from the group consisting of hydrocarbons and oil- derivatives, and synthetic compounds.

14. The use according to claim 13, wherein said synthetic compounds are selected from the group consisting of xenobiotics, pharmaceuticals, pesticides, phytosanitary compounds, herbicides, pesticides and dyes.

15. The use of any of claims 13 and 14, wherein the synthetic compounds are selected from the group consisting of the xenobiotic Bisphenol A, the pharmaceutical oseltamivir carboxylate and the herbicide atrazine.