The present invention relates to a method for growing biomass on sludge, with sludge including biological waste streams, like biological waste sludge, fish faeces, byproducts from food industries and algae. More specific the method grows biomass in the form of aquatic worms to produce biomass comprising specific compounds. Such specific compounds include fatty acids and amino acids, for example. Existing methods to grow biomass include the use of biological systems, like oily fish for producing omega-3 fatty acids, for example. As fish are only to a limited extent capable of producing these specific compounds, but mostly accumulate them from the food, these systems also accumulate toxic substances like mercury, dioxin, PCB' s, for example. Furthermore, natural fish stocks which could be used as feed or foodstuff are declining.

One object of the present invention is to improve the overall efficiency and sustainability of growing biomass comprising specific compounds.

This object is achieved with the method for growing biomass on sludge, comprising the steps of: providing aquatic worms in a reactor; providing a sludge to grow biomass comprising specific compounds; and supplying the reactor, comprising the worms, with sludge .

Worm biomass, especially the dry matter fraction of aquatic worm biomass, mainly consists of protein and small fractions of fat, herein also referred to as lipids, sugars, herein also referred to as carbohydrates, and ash. A reactor is provided with the aquatic worms. Preferably, a support carries the aquatic worms. Alternatively, worms are grown on a layer of sediment, for example. Examples of suitable carriers are carriers provided with openings (inclusive pores) or surfaces, wherein or whereon the worms may establish, flexible sponge-like materials, such as Recticel® or mesh-like materials. Preferably, a porous carrier is applied, having a mean pore size of 100 μm till several mm, such as 100-3000 μm, for example 200-1000 μm, more preferably 200-500 μm. For the processing of sludge, the aquatic worms, in particular those of the class of the Oligochaeta, are brought in a reactor wherein is provided a porous carrier, such as a net or cloth having a fine mesh, or a porous three dimensional carrier. In the openings (pores) of the porous carrier, the worms may nest and from there may feed on constituents of the sludge, which flows along and/or through the pores. Hereby the worm biomass in the porous carrier increases. The worm biomass produced according to the invention comprises compounds including proteins, lipids, fatty acids, carbohydrates, ash, and/or amino acids. Depending on the type of sludge, a specific type of aquatic worm produces specific compounds of biomass and/or increases the production of specific compounds thereby optimising the growing of biomass.

It has now surprisingly been found that when aquatic worms are grown on sludge as meant herein, the biomass of such aquatic worms comprises many polyunsaturated fatty acids (PUFA' s) as well as a specific amino acid profile which renders the worms particularly suitable for use as animal feed. For example, the amino acid composition sufficiently matches the requirements as set by the Committee on Animal Nutrition (Board on Agriculture,

National Research Council, 1993. Nutrient requirements of fish. National Academy Press, Washington DC, USA.) to render such worm mass particularly suitable for use as fish feed. Interestingly, when growing aquatic worms according to the invention, it was found that the amino acid composition remained stable when different sludge types were fed to the aquatic worms making them particularly suitable as fish feed. Worm biomass may be used in freeze-dried form, frozen form, as live animals, as fractions isolated from the worms (e.g. the lipid or protein fraction) or other suitable forms .

In contrast to the amino acid profile of the worm biomass which appears independent from the used type of sludge, after growing worm biomass on several types of sludge, it was observed that the composition of the sludge influences the composition of other groups of compounds of the worm biomass grown thereon. Consequently, inter alia, the lipid, fatty acid and carbohydrate contents of the worm biomass can be controlled or at least influenced using different types of sludge allowing one to produce animal feed or foodstuff suitable for certain purposes, thereby reducing the need for adjusting the amounts of certain components in particular cases. In particular, the percentages of compounds such as lipids, carbohydrates and ash were found to be variable with the type of sludge as used. With respect to the production of PUFA' s in the worm biomass, it was surprisingly found that several such compounds or groups of compounds were specifically concentrated in the worm biomass compared to the sludge source. The amount of several such compounds, e.g. ω-3 and ω-6 type PUFA' s, is increased in sludge grown worm biomass. A specific subgroup of PUFA' s, termed HUFA' s (highly unsaturated fatty acids) , show a marked overall concentration in worm biomass grown on sludge. This result is especially remarkable as SFA' s (saturated fatty acids) are reduced in sludge-grown worm biomass compared to SFA content of the worm-fed sludge.

Also, aquatic worms according to the invention, in particular L. variegatus, can accumulate bacterial fatty acids from their sludge food source. For example, worms grown on sludge comprising living bacteria contain 12 percent bacterial fatty acids, in contrast to worms grown on substrates with less bacterial biomass. Thus, additionally or alternatively, another object of the present invention is to provide a method for growing biomass on sludge, comprising the steps of:

- providing aquatic worms in a reactor;

- providing a sludge for growing aquatic worm biomass, and

- supplying the reactor, comprising the aquatic worms, with the sludge to allow aquatic worm biomass to grow, and

- obtaining specific compounds selected from the group consisting of proteins, lipids, fatty acids and amino acids from the aquatic worm biomass. In a preferred embodiment according to the present invention the specific compounds comprise protein, fatty acids, and/or amino acids.

The term "sludge" is meant herein in its broadest sense, meaning a biological residue in liquid to semi-solid form which is used as consumption feed for aquatic worms as meant herein. In particular, such sludge comprises byproducts of food industries, such as fish faeces from fish farms; sludges from the paper-processing industry; sludge from the sugar processing industry, in particular sugar beet or sugar cane; sludge from soy, starch, potato, cereal or dairy processing industry; wheat yeast concentrate; fruit and vegetable waste, in particular carrot peels; residues or by-products from the vegetable oil processing industry, in particular palm oil or soy-oil; algal sludge, in particular marine algal sludge; sludge from communal plants and/or any suitable mix of such products.

Protein constitutes the largest fraction of the dry matter of an aquatic worm like Lumbriculus variegatus and can, for example, be extracted under acidic or basic conditions followed by iso-electric precipitation. If this fraction is unpolluted, application as animal feed is an option. Other outlets for this protein could be technical applications like coatings, glues, emulsifiers, dispersion, foaming or wetting agents.

Amino acids have multiple applications. Separate amino acids are traditionally used as additives to animal feeds or as taste enhancer in human food. Arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine are essential amino acids that cannot be synthesized de novo by mammalian cells. Methionine and lysine are produced in the largest quantities, followed by threonine and tryptophan. These amino acids constitute respectively 2, 7, 6 and 1 % of L. variegatus protein.

Vegetable oils and animal fats are mainly applied in food (80 %) . The remainder is used for industrial applications. Poly-unsaturated oils, like linseed oil and soy oil, are used for manufacturing resins in paint and ink industries. Also, application of vegetable oils and animal fats as biodiesel is possible. For example, the fat fraction of L. variegatus, which can be obtained by rendering from the worm biomass, could be used for this purpose. Obtaining or isolating fatty acids can be achieved using any suitable means available to the skilled person, such as by supercritical fluid extraction according to Herrero et al, 2006. Fatty acids are mainly applied in the cosmetic (for example soaps and other surfactants) and lubricant industry. Other relevant applications of fatty acid derivatives are cleaning products, plastics and fabric softeners. There is an increasing interest in fatty acids as food additives or biofuels. Aquatic worms contain interesting fatty acids, such as the polyunsaturated (omega-3 and omega-6) , Eicosapentaenoic acid (EPA) , Docosahexaenoic acid (DHA) , Arachidonic acid (AA) , Linoleic acid (LA) , and Linolenic acid (ALA) . These compounds are also referred to as polyunsaturated fatty acids (PUFAs) . These essential fatty acids are very important for mammalian growth and development. Depending on the food source, the worm biomass can contain odd- and branched chain fatty acids (OBCFA, for example C15 and C17 fatty acids) of which at least some display anti-carcinogenic activity. The present invention thus also provides a method for the production of PUFA' s, HUFA' s and OBCFA' s by allowing worm biomass to grow on sludge as meant herein in a reactor. In a further preferred embodiment according to the present invention the sludge used as consumption feed for aquatic worms as meant herein, is in essence free of pollutants. In this embodiment, the sludge does not include communal waste derived-sludge as this may comprise pollutants in concentrations too high for use of the aquatic worms in subsequent feed or foodstuff applications. Although the worm mass grown on communal sludge comprises low amounts of heavy metals, which in fact are in the same range of concentrations in worms grown on commercial ornamental fish feeds, owing to strict local legislation and the possible contamination with other pollutants, it may not be allowed to use such biomass as consumption feed for animals or for human foodstuff purposes. Consequently, especially when grown on sludge which is in essence free of pollutants, the worm biomass may be used as a foodstuff for humans or animals. As it is estimated that already the Dutch food industries alone produce yearly at least 10 million tonnes of wet byproducts, this type of what is considered herein as pollutant-free sludge (or clean waste-stream) comprises an excellent consumption feed for obtaining worm biomass which can be used for further processing thereof into foodstuff for humans or feed and foodstuff for animals. According to the invention, aquatic worm biomass thus constitutes an alternative clean source of animal proteins, lipids and carbohydrates .

The sludge acts a feed stream to the aquatic worms. The inventors of the present invention have now found that aquatic worms are found to contain unusual fatty acids as part of the fatty acid composition and total content to vary as function of the composition and availability of the feed stream. The feed stream dictates appearance of the worms, fat, ash, lipid and fatty acid content and composition, and most likely also other biomass characteristics as well. In addition to and/or alternative to the sludges mentioned above the sludge may originate from other industrial sludges like those from soy, starch and dairy processing industries. In 2006 the Dutch feed- and drink industries produced 45 million kg dry waste sludge. Common disposal methods include use as fertilizer, composting, use as animal feed or incineration .

A further advantage when using sludge to grow biomass comprising specific compounds is the reduction of this sludge. Biological wastewater treatment plants (WWTPs) produce biological waste sludge (biosolids) , which is a complex mixture of water (up to more than 95 %) , bacteria, dead organic and inorganic materials, containing phosphorus and nitrogen components and various pollutants (e.g. heavy metals, organic pollutants and pathogens) . In Europe alone, more than 40,000 WWTP' s produce around 7 million tonnes of dry solids (DS) per year and this production is expected to increase, also on a global scale. In Europe, most sludges are settled, stabilised, thickened, anaerobically digested and then disposed of. Traditional disposal methods consist of application as agricultural fertilizer, disposal in landfills or the sea, or incineration. The costs of these treatment and disposal methods are high and estimated to be up to half of the operational costs of wastewater treatment. Heavy metal concentrations to an increasing extent give rise to problems in the first two disposal methods.

The biological method according to the invention, which addresses both the minimization of sludge production and the recovery of valuable components, is sludge reduction by aquatic worms. The consumption of sludge particles by worms not only leads to a decrease in the DS and volume of the sludge that has to be disposed of as worm faeces, but also to a conversion of part of the sludge into new worm biomass with potential for re-use because of, for example, its high protein content.

Experiments with aquatic worms show large variations for reduction percentages of the sludge (between 15 and 75 % of the dry matter) , depending on the experimental conditions. Also, the doubling time of aquatic worms, like L. variegatus on sludge can be as short as 7 days, which is relatively fast in comparison to those on other feeds like organic material in sediments (10-40 days) . In batch experiments, around 7 % of the total amount of sludge provided is converted into worm biomass, based on dry matter. A 100,000 p.e. (person equivalent) WWTP with a typical yearly waste sludge production of almost 2 kilotonnes DS (Statistics Netherlands (CBS), 2007) could thus produce 130 tonnes of worm dry weight (DW) , which equals 1 kilotonne of wet weight (WW) per year. Application of aquatic worms, like L. variegatus for both minimizing sludge production and recovering valuable sludge components therefore has high potential.

The basic composition of depurated (with empty guts) aquatic worms, like L. variegatus, grown on other feeds than sludge, i.e. fish feed or sediments, has been determined and is shown in Table 1.

Table 1: Biomass composition of depurated Lumbriculus variegatus (in % of DW) grown on fish feed or sediments. Worm DW was 15-16 % of WW.

Component % of DW

Protein 62-66

Fat 11-12

Sugar 13-18

Ash 9-11

Fatty acids 7-12

Calcium 0.2-0.3

Phosphorus 1.4-2.1

Calories (kcal/ g DW) 4.8-4.9

In a further preferred embodiment according to the present invention the aquatic worms are of the class of Oligochaeta .

Worms from the class of the Oligochaeta have shown to be capable to effectively produce biomass with specific compounds. Furthermore, they have shown to be extremely suitable for application in reducing and compacting sludge, which is produced in both communal and industrial waste water treatment plants, for example. Preferably, the aquatic worms are selected from the order of the Oligochaeta, more preferably from the family of the Lumbriculidae or the family of the Tubificidae, such as Nais variabilis or Nais simplex or Tubifex tubifex or the genus Limnodrilus, and most preferably the worms are selected from the genus Lumbriculus, such as the species Lumbriculus variegatus or from the genus Dero, for example the species Dero digitata. The species Lumbriculus variegatus has shown in experiments that it produces biomass with specific compounds effectively. Also, they exhibit a more stable growth on sludge than other worms, and moreover they replicate asexually. The latter aspect makes the processing of a predation reactor easier.

In a further preferred embodiment according to the present invention, separating means separate the waste sludge, worm faeces and worms.

The present invention thus also encompasses the step of obtaining or removing the aquatic worm biomass from the reactor by separating means. The removal of the worms from the reactor and separating from the support or carrier metal is important to enable an application of the worm biomass. A solution for the problem of this separation when using aquatic worms from the class of Oligochaeta, which are capable of motion by swimming, is inducing a so-called escape reflex. Such escape reflex is a neural physiological reaction which occurs in certain Oligochaeta in response to exposure to sub lethal concentrations of toxins or toxics. This escape reflex may be used to release the aquatic worms from the carrier material. Other separating means are also possible.

In order to prepare the worm biomass for consumption, the present invention further comprises the step of processing the worm biomass into consumption feed, in particular animal feed, or foodstuff suitable for human consumption. It lies within the capabilities of the average skilled person to prepare worm biomass into animal feed or foodstuff suitable for human consumption. In a particularly preferred embodiment of the invention, worm biomass may be used in freeze-dried or frozen form, as live animals or any other suitable form as animal feed for a variety of animals. The worm biomass may serve as main component of the animal feed or as a nutritional supplement comprised by the feed. In a further preferred embodiment according to the present invention the aquatic worms are used as test organism to be used in specific bioaccumulation and/or toxicity assays according to Ingersoll et al (2000).

As the inventors have found a relation between the specific compounds in the worm biomass, especially for the L. variegatus, and the sludge composition that is used as a feed stream to the worms, the worm biomass is an indicator for bioaccumulation and toxicity of the sludge. Furthermore, besides being used as indicator for the sludge the worms are also an indicator for the quality or efficiency of the operations producing these sludges.

The present invention also relates to the use of the grown biomass with the method according to the present invention as consumption fish feed. Based on the content of specific compounds in aquatic worms, especially L. variegatus, the aquatic worms are a good food source for several species of fish or other aquatic animals. Also, as an alternative the biomass is useful for ornamental fish food. Depending on the quality and the characteristics of the sludge as feed stream to the aquatic worms the grown biomass can also be applied as feed for consumption animals as long as the presence of heavy metals, organic micropollutants and pathogens which would end up in the human food chain is prevented.

The inventors of the present invention have now found that especially the fatty acid composition of the worm biomass depends on the fatty acid composition in the feed stream. Therefore, the use of sludge as a feed stream comprising a high concentration of PUFA' s will result in worm biomass also comprising a high content of PUFA' s . In addition, the use of sludge as a feed stream comprising a low concentration of PUFA' s will result in worm biomass comprising a higher content of PUFA' s . Examples of sludge that will result in appropriate biomass that can be used as fish feed, for example, are sludge from the production of Tilapia, and especially the faeces thereof, sludge from the sugar processing industry, sludge from the paper-producing industry, marine algal sludge, sludge from communal waste plant, carrot peels and wheat yeast concentrate. Therefore, the use of aquatic worms when growing biomass on a sludge provides an alternative for production of fish oil and fish meal that are often produced from wild fish like mackerel and salmon. Especially fish meal is interesting. Also, vegetable alternatives for fish oil and fish meal are often not completely effective, while, for example, the amino acids in L. variegatus are present in such ration that they fulfil all dietary requirements of fish for these compounds. The amount of fish that is available is decreasing, and, furthermore, may accumulate toxic substances. The use of aquatic worms according to the present invention prevents extension of these wild fish species. Furthermore, using sludge as a feed stream has the beneficial effect of sludge reduction, and, in addition, the recycling of variable raw materials in aquaculture and sludge processing. The present invention further also relates to a device for growing worm biomass, the device comprising: a reactor for the worms; aquatic worms provided on the reactor; - supply means for supplying sludge to the worms; and separating means to separate the aquatic worms, with specific compounds of biomass, from the sludge. Such device provides the same effect and advantages as those stated with reference to the method. Further advantages, features and details of the invention are elucidated on the basis of preferred embodiments thereof, wherein reference is made to the accompanying drawings, wherein:

- figure 1 illustrates a set-up for growing worm biomass; - figure 2A and B illustrates an alternative set-up for growing worm biomass;

- figure 3 shows experimental results showing cumulative amounts of added waste sludge and collected worm faeces from a continuous worm reactor; - figure 4 shows the components of amino acids in the biomass grown according to the invention;

- figure 5 shows the components of sugars in the biomass grown according to the invention; and

- figure 6A and B shows experimental results for heavy metals in the sludge and in the biomass.

- figure 7 shows FAs in worms and their feeds as percentage of total FAs. S is communal sludge, WS is worms grown on communal sludge, T is Tetra Min® ornamental fish feed, WT is worms grown on Tetra Min®, M is modified trout feed, WM is worms grown on trout feed. SAT is saturated FAs, HUFA is highly unsaturated FAs, ω-3 is total ω-3 FAs, ω-6 is total ω-6 FAs. - figure 8 shows an as yet unidentified poly-unsaturated fatty acid (boxed) in L. variegatus .

- figure 9 shows growth characteristics of L. variegatus grown on different types of sludge obtained as by- products from food industries. Wheat= wheat yeast concentrate, mix= mix of different by-products, carrot= carrot peels.

-figure 10 shows breakdown of and growth on different worm feeds in 7-day batch experiments. Fish faeces in this case is Tilapia fish faeces, Sugar sludge in this case is sludge from sugar-processing industry, Starch sludge in this case is sludge from starch-processing industry, TSS is total suspended solids, VSS is volatile suspended solids, # is worm number, ww is worm wet weight.

- figure 11 shows growth characteristics of L. variegatus on different marine algae. Tetraselmis = Tetraselmis suecica, Phaeodactylum = Phaeodactylum tricornutum, Neochloris = Neochloris oleoabudans . - figure 12 shows growth characteristics of L. variegatus grown on different types of sludge obtained from paper- producing industries. sec= secondary paper sludge, prim= primary paper sludge, combi= combination of primary and secondary paper sludge. In a system (figure 1) an aqueous waste water stream 4 is fed to a bioreactor 6 provided with a post-settling device, flotation device or membrane separation device 8. The water that is separated off by the separation devices leaves bioreactor 6 as an aqueous effluent stream 10. The excess sludge that is formed during the biological treatment is fed to a predation reactor 12 as waste sludge 14 and then predated in a predation reactor. In addition to or instead of the waste sludge from the bioreactor, sludge produced during pre-settling of an aqueous waste stream or sludge originating from a fermenter can also be fed to predation reactor 12. An oxygen-comprising water stream 16 is also fed to predation reactor 12, for which purpose aqueous effluent 10 from the bioreactor can optionally be used.

The stream leaves the reactor via outlet 18 and is removed or is recirculated, after it has passed through an aerator, as input 16 to predation reactor 12. The predated waste sludge 20 is removed or recirculated to the bioreactor 6. The effluent 22 from predation reactor 12 is therefore removed or recirculated to the bioreactor 6 or to the predation reactor 12. During the predation of waste sludge, the biomass of the aquatic worms increases. The increase is about 5% -20%, calculated on the basis of weight of the original amount of waste sludge and expressed in dry matter. The excess mass of worms is harvested and can, for example, appropriate be used in fish food and other aquatic organisms, and as a raw material for agricultural chemicals in adhesives, as a toxicity organism, in compositions comprising surface-active matter, in coatings, in biodegradable plastics, as a source of enzymes, as detergents, as a high-protein additive, or as a fertiliser, or is recirculated tot the bioreactor 6.

The waste sludge 14 is fed, along with the sessile worms, to the predation reactor 12, which is provided with a support 24 that preferably comprises a fine-mesh separation device, above the support 26. The waste sludge is predated by the worms in the support. The predated waste sludge 20 leaves the predation reactor 12 at the bottom of the support 24 and the effluent comprising non-predated sludge leaves the reactor at the top of the support 24. The support 24 therefore also has a separation function. The oxygen- comprising water 15 is fed to the bottom of the support 24 and also leaves the reactor via outlet 22 at the bottom thereof .

An alternative system 26 (figure 2) is configured according to the characteristics given in Table 2. Table 2: Dimensions of the worm reactor

mesh size carrier material μm 350 mesh cylinders # 3 surface area cm ² 1257 (x 3) height mesh cylinder Cm 100 diameter mesh cylinder Cm 4 volume sludge compartment L 1.3 volume water compartment L 31

Worms (29.8 g ww) can be introduced in the worm reactor 28 via the open top of the mesh cylinders 32. Waste sludge 34 from the activated sludge system 36, comprising a settler 38 and an aeration tank 40, is directly pumped to the inlet 42 of the sludge compartment, i.e. the bottom of the mesh cylinders 32. Effluent 44 from the activated sludge system 36 is collected in an overflowing bucket 46, from where it is pumped to the inlet of the water compartment. The effluent flow rate through the water compartment of the worm reactor 28 can be changed, for example, it can be decreased stepwise from 43 L/d to 2.8 L/d. Worm faeces 50 are pumped from the bottom of the water compartment at a rate of 1.2 L/d. The water compartment is aerated using a diffuser (not shown) , with an air flow rate of about 690 mL/min inside a pipe. This visibly creates some mixing of the effluent 46 in the water compartment, which could distribute dissolved oxygen throughout worm reactor 28, but at the same time allows worm faeces 50 to settle. The outflow 52 from the worm reactor 28 is collected and can be analyzed for total COD, soluble COD, ammonia, nitrate and phosphate, for example. In experiments, sludge that was not consumed by the worms was not found in the worm outlet, but formed a sludge bed inside the mesh cylinders. Collected worm faeces can be analyzed for TSS, total COD and its supernatant for total COD, ammonia, nitrate and phosphate, for example. In experiments, waste sludge and worm faeces were occasionally analyzed for total N and total P. In the experiments performed in system 26, at the end of the experimental run, all the worms in the mesh cylinders were collected and their ww was determined. Temperature and dissolved oxygen (DO) concentration in the water compartment of the worm reactor were measured using an optical dissolved oxygen measurement probe (Oxymax W C0S61, Endress and Hauser) (not shown) . Feasibility experiment A first experiment was performed with a small version of system 26 and Leeuwarden WWTP (communal) sludge to illustrate that worm biomass can be grown according to the invention on sludge. In a period of 40 days, the worm biomass in the reactor increased from 9.8 to 18 g ww. This showed that net worm growth rate (0.015 d ^"1 ) was possible also in this configuration, even higher than in a horizontal carrier (0.009-0.013 d ^"1 ) , but still below rates found for non-immobilized worms (0.026 d ^'1 ) .

A continuous worm reactor 28 was operated without any problems during the entire experimental period of nearly 8 weeks. The cumulative amounts of waste sludge fed to the worm reactor and collected worm faeces are shown in Figure 3

(cumulative TSS in gram for waste sludge in (filled 0) and worm faeces out (open 0) ) . In total 431 g TSS of waste sludge was fed to the worm reactor and 167 g TSS was collected as worm faeces. However, sludge accumulation was observed as a sludge bed in the mesh cylinders, which was expected since the reactor was started with an insufficient amount of worms. The amount of sludge consumed by the worms was therefore estimated from the amount of collected worm faeces and the TSS reduction (11 %) found in the batch experiments. This resulted in an estimated total sludge consumption of 187 g TSS and a total sludge digestion by the worms of 20 g TSS. The sludge consumption rate of 110 mg TSS/ (g ww-d) during the last days of operation, was lower than the 138 mg TSS/(g ww-d) in the sequencing batch experiment. This could be explained by the DO concentration of 6.7 mg/L in the water compartment, which was below the optimum concentration (8.1 mg/L) for the worms. Worm biomass

The worm reactor was started with 29.8 g ww of worms, divided over the three mesh cylinders. At the end of the 8 weeks of operation, 49.5 g ww of worms was found in the mesh cylinders. During operation of the worm reactor a total of 6.7 g ww of worms was collected with the worm faeces (worms that had fallen from the mesh) . Thus, a total worm growth of 26.8 g ww was observed, which corresponded with a yield of 0.2O g dw/g TSS digested by the worms. This is higher than the yield of 0.13 g dw/g TSS digested found in the continuous worm reactor with a horizontal carrier material, like the one shown in figure 1. The average worm net biomass growth rate was 0.014 d ^"1 , which is only slightly lower than the growth rate found in the feasibility experiment.

Visual inspection of the mesh cylinders showed that worms were situated along the entire sludge bed inside each mesh cylinder. By the end of the experiment the total sludge bed height in each cylinder had increased to 20-45 cm. However, most of the worms (~ 80 %) were situated in the top ~ 10 cm of the sludge bed. This corresponded to a worm density of 1.1 kg ww/m ² carrier material, which matched the stable worm density found in sequencing batch experiments with the same carrier material.

Experiment on biomass with specific compounds Experiments to illustrate that worm biomass comprising specific compounds can be grown according to the invention, are performed on a system similar to system 2 using non- immobilized L. variegatus cultures that originated from commercially available ^λ Tubifex' mixtures (pet shops) . They were maintained in an artificial ditch in a laboratory, which was constantly fed with effluent and sludge particles from a lab-scale activated sludge system treating wastewater from the municipal WWTP of the village of Bennekom. For comparison, also L. variegatus fed with sludge from the municipal WWTP of the city of Leeuwarden were used for heavy metal analyses.

Dry weight (DW) of the worms was determined after drying overnight at 105 ⁰ C and ash content after overnight ignition at 525 ⁰ C. DS of the sludge were determined according to standard methods known to the skilled person using black ribbon filters (12-25 μm, Schleicher and Schuell) .

For Protein analysis dry and milled worm material (20- 50 mg protein) was put in a Kjeldahl tube to which 1 Kjeltab and 9 mL of concentrated sulphuric acid were added. Destruction was performed for 50 minutes at 420 ⁰ C in a

Gerhardt Kjeldatherm apparatus. After 10 minutes of cooling, 75 mL water was added. Subsequently stream distillation using Gerhardt Vapodist was performed for 4.5 minutes. Finally, the nitrogen content was determined using titration with 0.1 M HCl. Protein amount was calculated using a

Kjeldahl factor of 6.25. Protein in sludge was measured by the Biuret method. Molecular weight distribution of the protein fraction was determined by gel electrophoresis (SDS-PAGE) . SDS-PAGE was carried out with 15 % polyacrylamide gel. Samples (10 mg protein) were mixed with 600 μL sample buffer with B- mercaptoethanol, heated at 90 ⁰ C for 5 minutes and centrifuged. The samples (10 μL) were applied on the gel. The gels were stained with Coomassie brilliant blue.

Fat was determined by Soxhlet extraction with hexane . The samples were extracted with soxtec-extraction using hexane at boiling temperature for 30 minutes and then washed with hexane during 75 minutes at room temperature. The extracted samples were allowed to dry at 60 ⁰ C during 16 hours. The weight of the samples was measured before and after extraction. For sugar analysis the milled samples were extracted with soxtec-extraction using ethanol : toluene 2:1, 96 % (v/v) ethanol and hot water (1 hour) at boiling temperature. The extracted samples were dried at 60 ⁰ C for 16 hours. The content of neutral sugars of the ethanol-extracted material was determined after a two-step hydrolysis with sulfuric acid (12 M for 1 hour at 30 ⁰ C; 1 M for 3 hours at 100 ⁰ C) according to modified TAPPI methods. Neutral sugars were determined by HPAEC with pulsed amperometric detection on a CarboPac PAl column (Dionex) with a water-sodium hydroxide gradient. The total sugar content of sludge was determined by the phenol sulphuric acid method with glucose as a standard.

For amino acid analysis, to dry worm samples (about 1 mg protein) 300-500 μl 6 M HCl was added and hydrolysis of the protein took place during 24 hours at 100 ⁰ C. After centrifugation, about 500 μL 20 mM HCl was added in order to get a concentration of about 0.2 mg/mL. The amino acids were derivatised with AccQ. Flour reagens . 5 μL of the obtained solution was injected in a HPLC having a Nova-Pak™C18 column. The eluens was a 40/60 water/acetonitril mixture. The column temperature was 30 ⁰ C, the flow rate was 1 mL/min. Identification of the amino acids took place based on the retention times.

Using this method however, tryptophan is being destroyed. Therefore, tryptophan was determined separately by Ansynth Service BV (Roosendaal, the Netherlands) .

Total nitrogen and total phosphorus were determined according to Standard Methods known to the skilled person, using Dr Lange ^® test tubes.

For determining the heavy metal concentrations in L. variegatus, two long-term experiments were performed. In the first experiment, L. variegatus cultures were grown on sludge from municipal WWTP Bennekom, the Netherlands, for six months. As control, a L. variegatus culture was grown on Tetra Min ^® fish feed (for tropical fish) during the same period. According to the label, the fish feed contained 49 % protein, 9 % fat, 2 % cellulose and 12 % ash (DS based) plus added vitamins A, D3 and E. The cultures were fed weekly in excess. After six months, Cd, Cr, Cu, Ni, Pb and Zn were extracted from the worms, the control worms, the sludge and the fish feed by a microwave assisted aqua regia destruction step. Destruates were filled up to 100 mL with milliQ and filtered. 1 mL from each solution was dissolved in 9 mL milliQ and then analysed on an ICP-MS (0.14 M HNO ₃ matrix) by a commercial laboratory (Soil Chemical and Biological Laboratory, Wageningen, the Netherlands) .

In the second experiment, L. variegatus cultures were grown on sludges from municipal WWTPs Bennekom and

Leeuwarden, the Netherlands, for five months. The cultures were fed weekly in excess. After five months, As, Cd, Cu, Cr, Pb, Hg, Ni and Zn in the two worm cultures and the two sludges were extracted and analyzed by the same laboratory as in the first experiment.

Specimens of L. variegatus grown on sludge generally are larger (up to 45 mg) than those grown on other feeds like sediments or fish feed (typically 5-10 mg) .

This indicates that sludge has a very high nutritional value. Individual wet weight increased in feeds with higher organic material content, while reproduction rates remained the same. It was also observed that the tissue colour of L. variegatus grown on sludge is dark red, while that of worms fed on fish feed is pink.

The main components of L. variegatus biomass grown on sludge are Protein, Fat, Sugar, Ash, Fatty acids, Calcium, Phosphorus and Calories, see Table 3. Table 3: Main components of depurated L. variegatus (in % of DW) and the sludge from WWTP Bennekom used to grow the worms (in % of DS) . Worm DW was around 13 % of the WW.

Component Worms Sludge

% of DW % of DS

Protein 63 34-43

Fat 25 nd*

Sugar η * * 23-26

Ash 6 14-22

Phosphorus 0.9-2.2 1.6-1.7

Nitrogen 11-13 6-10

* Fat was not determined in the sludge but constituted most likely the major part of the missing DW fraction (19-25 %) , which also contained other components like humic acids, bacterial DNA and RNA. ** Sugar content was calculated

Results for amino acids and sugar are presented in separate figures (Figure 4 as % of total amino acids, and Figure 5 as % of total sugars (monosaccharide) ) . Sugar and ash content were somewhat lower in L. variegatus grown on sludge compared to worms on fish feed, while the fat content was twice as high. A higher fat content can indicate a higher nutritional value of the feed. Although sludge and fish feed have a similar basic composition, with exception of living bacteria, surprisingly, sludge appeared to be more nutritious than fish feed. Most results for L. variegatus grown on sludge, except for the fat content, were also similar to those for other aquatic Oligochaeta.

Typical values for protein content in activated sludge are rather stable (32-41 %) and comparable to what we found, but those for ash and sugar content are variable, respectively 12-41 % and 10-45 %) of the DS. In comparison to the feed sludge, L. variegatus biomass is significantly enriched in protein and (naturally) nitrogen, but contained lower concentrations of ash and sugar. Fat and phosphorus concentrations were comparable.

The proteins isolated from L. variegatus have a broad molecular weight distribution varying from 10 kD to 300 kD. Some protein fractions were found with a very high molecular weight. However, the major part of the protein had a molecular weight between 14 and 20 kD under reduced conditions . The amino acid composition was comparable to that described for L. variegatus grown on fish feed, with high percentages of alanine, aspartic acid, glutamic acid, glycine, leucine and lysine. In contrast, in the present experiment the presence of asparagine, cysteine and glutamine was found, while in the current research no cystine was found. However, during the analysis process, these amino acids can be easily converted into aspartic acid, cystine and glutamic acid respectively, which may explain the different results. Again, the results were similar to those for other aquatic Oligochaeta, for example T. tubifex.

The heavy metal concentrations in L. variegatus grown on different sludges and a control feed (Tetra Min ^® fish feed) from two long-term experiments are shown in Figure 6 in mg/kg DW or DS for substrate (open bars is substrate with B is sludge and F is fish food, and filled bars is worms) . L. variegatus is capable of accumulating heavy metals in high concentrations. Clearly however, in both experiments the concentrations of heavy metals in L. variegatus grown on sludge for long periods remained usually below those in sludge. Only Cd and Zn in Experiment 1 were found in similar concentrations in sludge and worms. The low bioaccumulation may result from binding of the metals to the organic fraction of the sludge (57-66 %) , which is much larger than that of sediments (typically a few percent) . In analogy, Tubificidae are known to bioaccumulate heavy metals, dependent on environmental conditions like organic matter concentrations. However, similar to Tubificidae, L. variegatus almost exclusively digests the organic fraction of the sludge (which contains most metals) and most likely regulates metal uptake. Tubificidae are known to possess detoxification mechanisms for metals like internal compartmentalization and binding to metallothionein- proteins. These proteins possibly are also involved in excretion of the metals. In support of this, the metal concentrations in the worms in both experiments were independent of the concentrations in the feeds (sludge or fish feed) . This was especially obvious in Experiment 1 for Cu and Zn (Figure 6) .

Experiments with use of different types of sludge In a first experiment reduction of and growth on waste sludge (fish faeces) of Tilapia is tested. Worms were fed with fish faeces (washed with demiwater) . The worms were able to reduce the faeces with ~ 30 % and compact them into worm faeces (higher settleability) . Furthermore their growth yield was 0.24 (mg dry weight worm produced/ mg dry weight faeces digested) . In total, 7 % of the fish faeces were converted into new worm biomass (dry weight based) .

In a second experiment reduction of and growth on waste sludge from sugar-processing industries and paper-producing industries is tested. Worms were fed with secondary sludge from a sugar-processing industry (washed with demiwater) . The worms were able to reduce the sludge with ~ 20 % and compact it into worm faeces (higher settling ability) . Furthermore their growth yield was 0.46 (mg dry weight worm produced/ mg dry weight sludge digested) . In total, 8 % of the sugar sludge was converted into new worm biomass (dry weight based) . In addition worms were fed with secondary, primary and combined sludges from a paper-producing industry and biomass growth was seen in all cases (figure 12) .

In a third experiment fatty acid profiles of worms grown on municipal sludge and Tetra Min fish food were measured. Worms were grown on municipal sludge or Tetra Min ^® fish food for more than 6 months. Fatty acid profiles of both populations were determined (Table 4).

Data were used from manufacturers, Hansen et al . (2004) and Elissen et al . (2010) . Additional analyses were done by Analytico BV (the Netherlands) and according to the lipid analysis by hexane/acetone extraction described in Jonker et al. (2009) .

Fatty acid analysis

Three worm populations and their feeds as described above (sludge from WWTP Leeuwarden, Tetra Min® and modified trout feed) were analyzed by Analytico (Heerenveen, the Netherlands) . The lipid fraction was extracted with acetone/petroleumether and dissolved in hexane . The FAs were methylated with alcoholic KOH and the extract was analyzed on a GC with flame ionization detector. Results are shown in Figure 7.

Table 4 : fatty acid profiles

Sludge 22/1/9 Tetra Min 4/2/9

% of total % of total

Full name det <0 1 det <0 3

C12 0 Launnezuur 0 6

C13 0 Tridecaanzuur 0 2

C14 0-ιso 12-Methyltrιdecaanzuur 0 6

C14 0 Mynstinezuur 2 2 24

C14 1 Tetradeceenzuur 0 3

C15 0-ιso 13-Methyltetradecaanzuur 44 0 5

C15 0-ante-ιso 12-Methyltetradecaanzuur 0 9

C15 0 Pentadecaanzuur 04

C16 0-ιso 14-Methylpentadecaanzuur 0 6

C16 0 Palmιtιnezuur 4 9 84

C16 1 7-Hexadeceenzuur 1 0 1 0

C16 1 9-Hexadeceenzuur 4 2 1 5

C16 2 9,12-Hexadecadιeenzuur 0 2

C17 0-ιso 15-Methylhexadecaanzuur 1 7

C17 0-ante-ιso 14-Methylhexadecaanzuur 1 6

C17 0 Margannezuur 1 2 0 7

C17 1 9-Heptadeceenzuur 0 2

C18 0 Steannezuur 64 7 1

C18 1 Ohezuur (incl cis-isomeren) 10 6 14 2

C18 2 Lιnolzuur 24 6 2

C18 2 Ovenge cis-isomeren 0 8 0 3

C18 3 Linoleenzuur 04 1 2

C20 0 Arachinezuur 0 2 04

C20 1 Eicoseenzuur 0 5 1 3

C20 2 Eicosadieenzuur 3 8 8 8

C20 3 8,11,14-Eιcosatrιeenzuur 1 6 1 1

C20 3 11,14,17-Eιcosatrιeenzuur 0 3 0 6

C204 Arachidonzuur 6 8 6 9

C204 8, 11 , 14, 17-Eιcosatetraeenzuur 04 04

C20 5 5,8, 11 , 14, 17-Eιcosapentaeenzuur 6 1 11 5

C21 0 Heneicosaanzuur 0 3

C22 0 Beheenzuur 0 2 0 5

C22 1 Cetoleinezuur 0 2 04

C22 5 7,10,13,16,19-Docosapentaeenzuur 0 7 2 0

C22 6 Docosahexaeenzuur 0 7 54

C23 0 Tricosaanzuur 0 1

C: Unknown 30.4 8.3

TOTAL 97.9 91.1

Cis-Mono unsaturated fatty acid 17.0 18.3

Cis-Poly unsaturatedfatty acids 24.3 44.4

Saturated fatty acids 26.6 20.7

Sum of C18:1 trans-isomers 1.3 1.7

Sum of C18:2 trans-isomers 0.4

Sum of C18:3 trans-isomers 6.5

Sum of trans-fatty acids 1.6 8.2

Sum of omega-3 fatty acids 8.6 21.1

Sum of omega-6 fatty acids 14.7 23.0

As is clear from the results shown in figure 7, the food source determined the fatty acid profile. Worms grown on fish food contained higher concentrations of HUFAs (e.g. 11.5 and 5.4 % of the total FA were EPA and DHA respectively in the worms grown on fish food, while these concentrations were 6.1 and 0.7 % respectively in the worms grown on sludge), ω-3 and ω-6 FAs constitute respectively 8.6 and 14.7 % of total FAs in worms grown on sludge and 21.1 and 23.0 % of total FAs in worms grown on Tetra Min ^® fish food. Results are shown in Figure 7. Figure 8 shows a thus far unidentified poly-unsaturated fatty acid obtained during these experiments.

Batch experiments were done according to the method described in Buys et al . (2008), wherein L. variegatus was fed with Tilapia faeces obtained from the Aquaculture and Fisheries Department (Wageningen University and Research Centre, the Netherlands) and secondary waste sludges from a sugar-processing (Suiker Unie, the Netherlands) and a starch-processing industry (AVEBE, the Netherlands) .

Secondary sludge mainly consists of bacteria and organic materials and originates from the aeration tanks of wastewater treatment plants. The sludge samples were always stored at 4 ⁰ C until use. Table 8 shows the main characteristics of these worm feeds. The biochemical composition of the feeds was unknown.

Table 5. More detailed analysis of FA profiles of three different worm feeds (% of total FAs) and three different worm populations. SFAs saturated FAs; MUFAs monounsaturated FAs (1 double bond); PUFAs polyunsaturated FAs (2, 3 or 4 double bonds) ; HUFAs highly unsaturated FAs (5 or 6 double bonds) . Empty cells indicate measurements below detection limits. Detection limits of the samples, based on the lipid percentage, were * 0.05 %, ** 0.10 % and *** 0.30 %.

Sludge Tetra Modified Worms Worms Worms

Min®** trout sludge** Tetra Min® trout feed feed* _{** *}

SFAs:

C4:θ O.65 C6:θ 0.06 C8:O 0.23

CiO: O 0.36 0.08 0.05

Cn: O # 1-37

Ci2:o 1.26 0.14 0-59 0.41

Ci3:θ # 0.60 0.15

Ci4:θ iso # 0-35 0.58 0.07

Ci4:o 2.06 3-47 2.92 2.18 2.40 2.63

Ci5:o iso # 1.01 0.11 0.16 4-39 0.51 0.70

Ci5:O ante iso # 0.56 0.17 0.90 0.26

Ci5:o # 0-45 0.28 0-57 0.44 0.50

Ci6:O iso # 0.15 0.15 0.62 0.16

Ci6:o 29.40 14.70 23-6 4.90 8.42 9-94

Ci7:θ iso # 0.18 0.10 0.31 1.71 0.63

Ci7:O ante iso # 1.58

Ci7:o # 0-35 0.31 1.07 1-15 0.71 1-39

Ci8:o 26.00 8-73 14.4 6-43 7.08 9-32

Ci9:θ # 0.11 0.24 0.15

C2θ:θ 0.48 2.15 0.22 0.23 0-37 0.31

C2i:o # 0.10 0.07 0.31 0-54

C22:θ 0.82 2.41 0.11 0.15 0.46 0-35

C23:o # 0.26 0.06 0.11

C24:o 0.16 0.26 0.10 0.13

2 SFAs 66.44 32.99 44 37 26.42 19 95 27.60

MUFAs:

Ci4:i 0.22 0.25

Ci5=i 0.27

Ci6:i 7 0.67 0.29 1.02 0.99 2.04

Ci6:i 9 3-26 2.81 2-55 4-23 1.50 2.84

Ci7:i 9 # 0.11 0-43 0.16 0.27

Ci8:i (incl cis) 8.52 21.10 29.90 10.60 14.20 13-50

C2θ:i eico 0.18 2.12 0-35 0.46 1-33 1.72

C22:i eru 0.31 0.07

C22:i ceto? 2.04 0.21 0-35 0.21

C24:i 0.28 0.16

∑ MUFAs 12.63 28.77 33 47 16.93 18.37 20.92

PUFAs:

Ci6:2 9, 12 0.31 0.20 0.13

Ci6:3 6, 9, 12 0.32 0.60

Ci8:2 ^« LA 3-95 19.40 9.01 2-43 6.20 5-30

Ci8:2 cis 0.12 0.21 0.08 0.77 0.31 0.78

Ci8:3 - ALA 0.69 2.62 1.24 0.41 1.22 0.66

Ci8:3 6, 9, 12 ^« 0.15

C20:2 " 0.21 0.40 0.08 3-83 8.84 6.88

C2θ:3 8, 11, 14 * 1-59 1.11 1.16

C2θ:3 11, 14, 17 - 0.18 0.06 0.25 0.62 0.44

2 PUFAs 5 12 23 44 1O.47 9 48 18.30 15 95

HUFAs:

Ci6:44, 7, 10, 13 - 0.38

Ci8:4 6, 9, 12, 15 ^~ 0.68 0.12

C2θ:4 ^« AA 0-59 0.38 0.38 6-75 6.87 4-93

C2θ:4 8, 11, 14, 17 - 0.48 0.08 0-39 0.40 0.22

C2O:5 - EPA 0.81 4-35 0.78 6.12 11.50 4-53

C22:5 - 1-15 0.23 0.65 1.96 0.49

C22:6 - DHA 0.16 4.12 4-35 0.71 5-43 1.79

2 HUFAs 1.56 11-54 5 94 14.62 26.16 11.96

2 unknown FAs 13 00 2.40 1.46 30.40 8.34 22.80

Sludge Tetra Modified Worms Worms Worms

Min®** trout sludge** Tetra Min® trout feed* feed* _{** *}

2 total FAs 98.75 99 14 95 70 97 85 91.12 99 23

∑ ω-3 ^~ 1.66 13.90 6.86 8.61 21.10 8.13

∑ ω-6 ^« 4.90 20.40 9 59 14 70 23.00 18.30 ω-3:ω-6 ratio O.34 O.68 0.44 O.59 0.92 0.44 Bacterial FAs # 5 0 1-3 2.8 11.9 1.2 4.4

Table 6. Spearman's rho correlations between FA profiles of three different worm feeds and three different worm populations.

Worms Worms Sludge Tetra Min® Modified

Tetra Min® trout feed trout feed

Worms 0.753 0.808 0.525 0.404 0.641 sludge

Worms 0.845 0.498 0.722 0.632 Tetra Min®

Worms 0.505 0.604 0.700 trout feed Sludge 0.297* 0.539 Tetra Min® 0.611

All correlations are significant at the 0.01 level (2- tailed) except * = significant at the 0.05 level (2-tailed) .

Table 7. Biochemical composition of worms and their feeds. Composition of fish feeds from manufacturer, composition of sludge and worms grown on sludge from Elissen et al . (2010), composition of worms grown on Tetra Min® from Hansen et al . (2004) and own research, composition of worms on trout feed analyzed by Analytico BV, the Netherlands. Missing percentages for lipid (*) or carbohydrate (**) were always calculated as 100 % = protein + lipid + carbohydrate + fibre + ash, na = not analyzed. Worms grown on sludge and Tetra Min were additionally analyzed for lipid content according to the method described by Jonker et al, 2009.

Sludge Tetra Modified Worms Worms Worms

Min® trout sludge Tetra Min® trout feed feed

Protein 34-43 50 38 63 62-66 64

Lipid 19-25* 11 7 7-25 11-17 8

Carbohydrate 23-26 24** 45 ^** 6-24** 13-18** 22**

Ash 14-22 12 8 6 9-11 6

Fibre na 3 2 na na na

In the experiments using marine algae, samples (10-100 mL) of marine algae (Tetraselmis suecica, Neochloris oleoabundans, Phaeodactylum tricornutum) were washed by centrifugation and addition of tapwater. Subsequently, they were added to glass Petri dishes containing populations of L. variegatus (0.1-0.5 g wet weight) and 600-800 mL of tapwater. Worm biomass was weighed weekly. Results are shown in Figure 11. In the same setup, byproducts from food industries (0.1-1OmL) were tested (carrot peels, wheat yeast concentrate, mix of several byproducts) and) . Results are shown in Figure 9. Also, sludges from the paper-producing industry, such as secondary, primary and combined sludge were tested. Results are shown in figure 12.

Table 8 Main characteristics of worm feeds used for batch experiments. TSS= total suspended solids

TSS g/kg Ash % pH Total (and unionized) ammonia mg/L

Tilαpiα faeces 42 15 6.4 74-5 (0.074) Sugar sludge 16 42 7-1 30.0 (0.149) Starch sludge 5 13 6.8 2.6 (o.oo6)

The present invention is by no means limited to the above described embodiments thereof. The rights sought are defined by the following claims, within the scope of which many modifications can be envisaged.

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