NL2002937C2

NL2002937C2 - METHOD AND DEVICE FOR GROWING BIOMASS ON SLUDGE.

Info

Publication number: NL2002937C2
Application number: NL2002937A
Authority: NL
Inventors: Hellen Johannes Hubertina Elissen
Original assignee: Stichting Wetsus Ct Excellence Sustainable Water Technology
Priority date: 2009-05-27
Filing date: 2009-05-27
Publication date: 2010-11-30
Also published as: WO2010137980A1; EP2440499A1

Description

METHOD AND DEVICE FOR GROWING BIOMASS ON SLUDGE

The present invention relates to a method for growing biomass on sludge, with sludge including biological 5 waste streams, like biological waste sludge, fish faeces 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.

10 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 very limited extent capable of producing these specific compounds, but mostly accumulate them from the food, these systems also 15 accumulate toxic substances like mercury, dioxin, PCB's, for example .

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

20 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 25 - 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, sugar and ash. A reactor is provided with 30 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 2 surfaces, wherein or whereon the worms may establish, flexible spongelike materials, such as Recticel® or mashlike materials. Preferably, a porous carrier is applied, having a mean pore size of 100 pm till several mm, such as 100-3000 5 pm, for example 200-1000 pm, more preferably 200-500 pm. For this application sludge specifically includes biological waste streams, like biological waste sludge, fish faeces and algae .

For the processing of sludge, the aquatic worms, for 10 example 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 15 the sludge, which flows along and/or through the pores. Hereby the worm biomass in the porous carrier increases.

The biomass produced according to the invention comprises compounds including proteins, fat, sugar, ash, and/or amino acids. Depending on the type of sludge a 20 specific type of aquatic worm produces specific compounds of biomass and/or increases the production of specific compounds thereby optimising the growing of biomass.

In a preferred embodiment according to the present invention the specific compounds comprise protein, fatty 25 acids, and/or amino acids.

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 30 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.

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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, 5 methionine, phenylalanine, threonine, tryptophan, tyrosine and valine are essential amino acids that can not 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 10 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 15 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 the worm biomass, could be used for this purpose.

Fatty acids are mainly applied in the cosmetic (for 20 example soaps and other surfactants) and lubricant industry. Other relevant applications of fatty acid derivatives are cleaning products, plastics and fabric softeners. Aquatic worms contain interesting fatty acids, such as the polyunsaturated (omega-3 and omega-6), Eicosapentaenoic acid 25 (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 30 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 disply anti-carcinogenic activity.

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In a further preferred embodiment according to the present invention the sludge originates from fish production, sugar processing, and/or communal sludge plants.

The sludge acts a feed stream to the aquatic worms.

5 The inventors of the present invention have now found that aquatic worms are found to contain unusual fatty acids with 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, fat content, 10 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 soy, starch and dairy processing industries. In 2006 the Dutch feed- and 15 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 20 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 25 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 30 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 5 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, 5 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 10 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 % 15 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 20 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 25 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.

30 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.

β

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 5 In a further preferred embodiment according to the present invention the aguatic 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 10 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 aguatic worms are selected from the 15 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, and most preferably the worms are selected from the genus Lumbriculus, such as the species Lumbriculus variegatus or from the genus Dero, for 20 example the species Dero digit at a. 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 7 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 5 sludge, worm faeces and worms.

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 10 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 15 be used to release the aquatic worms from the carrier material. Other separating means are also possible.

In a further preferred embodiment according to the present invention the aquatic worms are used as test organism to detect specific bioaccumulation and/or toxicity 20 assays.

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 25 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 30 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 8 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 5 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 10 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.

15 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 communal waste plant. Therefore, the use of aquatic 20 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 25 completely effective, while, for example, the amino acids in L. Variegates 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 30 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 9 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: 5 - 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.

10 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 15 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 20 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 25 grown according to the invention; and figure 6A and B shows experimental results for components in the sludge and in the biomass.

In a system (figure 1) an aqueous waste water stream 4 is fed to a bioreactor 6 provided with a post-settling 30 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 10 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 5 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 10 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 15 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, 20 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 25 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 30 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 11 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 5 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 pm 350 mesh cylinders # 3 surface area cm2 1257 (x 3) height mesh cylinder Cm 100 diameter mesh cylinder Cm 4 volume sludge compartment L 1.3 volume water compartment L 31 10 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 15 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 20 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 25 the water compartment, which could distribute dissolved oxygen throughout worm reactor 28, but at the same time 12 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 5 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 10 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 15 were measured using an optical dissolved oxygen measurement probe (Oxymax W COS61, Endress and Hauser) (not shown).

Feasibility experiment A first experiment was performed with a small version 20 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 25 also in this configuration, even higher than in a horizontal carrier (0.009-0.013 d-1), but still below rates found for non-immobilised worms (0.026 d-1).

A continuous worm reactor 28 was operated without any problems during the entire experimental period of nearly 8 30 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 13 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 5 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 10 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 15 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 20 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 25 0.20 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 30 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 14 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/m2 carrier material, which matched the 5 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 10 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 1Tubifex' mixtures (pet shops). They were maintained in an artificial ditch in a laboratory, 15 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 20 metal analyses.

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

30 Destruction was performed for 50 minutes at 420 °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.

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Finally, the nitrogen content was determined using titration with 0.1 M HC1. Protein amount was calculated using a Kjeldahl factor of 6.25. Protein in sludge was measured by the Biuret method.

5 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 pL sample buffer with B-mercaptoethanol, heated at 90 °C for 5 minutes and 10 centrifuged. The samples (10 pL) 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 15 with hexane during 75 minutes at room temperature. The extracted samples were allowed to dry at 60 °C during 16 hours. The weight of the samples was measured before and after extraction.

For sugar analysis the milled samples were extracted 20 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 °C for 16 hours. The content of neutral sugars of the ethanol-extracted material was determined after a two-step hydrolysis with sulfuric 25 acid (12 M for 1 hour at 30 °C; 1 M for 3 hours at 100 °C) according to modified TAPPI methods. Neutral sugars were determined by HPAEC with pulsed amperometric detection on a CarboPac PA1 column (Dionex) with a water-sodium hydroxide gradient. The total sugar content of sludge was determined 30 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 pi 6 M HC1 was added and hydrolysis of 16 the protein took place during 24 hours at 100 °C. After centrifugation, about 500 pL 20 mM HC1 was added in order to get a concentration of about 0.2 mg/mL. The amino acids were derivatised with AccQ.Flour reagens. 5 pL of the obtained 5 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 °C, the flow rate was 1 mL/min. Identification of the amino acids took place based on the retention times.

10 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, 15 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 20 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 25 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

30 milliQ and then analysed on an ICP-MS (0.14 M HN03 matrix) by a commercial laboratory (Soil Chemical and Biological Laboratory, Wageningen, the Netherlands).

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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, 5 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 10 like sediments or fish feed (typically 5-10 mg) (Figure 4). 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. Figure 4 also surprisingly shows that the tissue 15 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.

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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 7** 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 5 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 10 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, 15 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 20 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, 25 but those for ash and sugar content are variable, 19 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 5 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 10 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, 15 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 20 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 Γ. tubifex.

The heavy metal concentrations in L. variegatus grown 25 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=substrate with B=sludge and F=fish food, and filled bars=worms). L. variegatus is capable of accumulating heavy metals in high 30 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 20 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, 5 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 10 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 15 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).

20 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 25 worm faeces (higher settleability). Furthermore their growh 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 30 sludge from sugar-processing 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 21 faeces (higher settleability). Furthermore their growh 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).

5 In a third experiment fatty acid profiles of worms grown on municpal 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).

10 22

Table 4: fatty acid profiles

Slib 22/1/9 TetraMin 4/2/9 % of total % of total

Full name det<0.1 det<0.3 C12:0 Laurinezuur 0.6 C13:0 Tridecaanzuur 0.2 C14:0-iso 12-Methyltridecaanzuur 0.6 C14:0 Myristinezuur 2.2 2.4 014:1 Tetradeceenzuur 0.3 C15:0-iso 13-Methyltetradecaanzuur 4.4 0.5 C15:0-ante-iso 12-Methyltetradecaanzuur 0.9 C15:0 Pentadecaanzuur 0.4 C16:0-iso 14-Methylpentadecaanzuur 0.6 016:0 Palmitinezuur 4.9 8.4 016:1 7-Hexadeceenzuur 1.0 1.0 016:1 9-Hexadeceenzuur 4.2 1.5 016:2 9,12-Hexadecadieenzuur 0.2 017:0-iso 15-Methylhexadecaanzuur 1.7 017:0-ante-iso 14-Methylhexadecaanzuur 1.6 017:0 Margarinezuur 1.2 0.7 017:1 9-Heptadeceenzuur 0.2 018:0 Stearinezuur 6.4 7.1 018:1 Oliezuur (incl. cis-isomeren) 10.6 14.2 018:2 Linolzuur 2.4 6.2 018:2 Overige cis-isomeren 0.8 0.3 018:3 Linoleenzuur 0.4 1.2 020:0 Arachinezuur 0.2 0.4 020:1 Eicoseenzuur 0.5 1.3 020:2 Eicosadieenzuur 3.8 8.8 020:3 8,11,14-Eicosatrieenzuur 1.6 1.1 020:311,14,17-Eicosatrieenzuur 0.3 0.6 020:4 Arachidonzuur 6.8 6.9 020:48,11,14,17-Eicosatetraeenzuur 0.4 0.4 020:5 5,8,11,14,17-Eicosapentaeenzuur 6.1 11.5 021:0 Heneicosaanzuur 0.3 022:0 Beheenzuur 0.2 0.5 022:1 Cetolei'nezuur 0.2 0.4 022:5 7,10,13,16,19-Docosapentaeenzuur 0.7 2.0 022:6 Docosahexaeenzuur 0.7 5.4 023:0 Tricosaanzuur 0.1 C: Onbekend 30.4 8.3 TOTAL 97.9 91.1

Cis-Enkelv. onverzadigde vetzuren 17.0 18.3

Cis-Meerv. onverzadigde vetzuren 24.3 44.4

Verzadigde vetzuren 26.6 20.7

Som van C18:1 trans-isomeren 1.3 1.7

Som van C18:2 trans-isomeren 0.4

Som van C18:3 trans-isomeren 6.5

Som van trans-vetzuren 1.6 8.2

Som van de omega-3 vetzuren 8.6 21.1

Som van de omega-6 vetzuren 14.7 23.0

Clearly, the food source determined the fatty acid profile. 5 Worms grown on fish food contained higher concentrations of PUFAs (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-β FAs constitute respectively 10 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.

23

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.

24

Clauses 1. Method for growing biomass on sludge, comprising the steps of : 5 - providing aquatic worms in a reactor; providing a sludge to grow biomass comprising specific compounds; and supplying the reactor, comprising the worms, with sludge.

10 2. Method according to clause 1, wherein the specific compounds comprises protein, fatty acids, and/or amino acids .

15 3. Method according to clause 2, wherein the fatty acids comprise polyunsaturated omega-3 and omega-6 EPA, DHA, AA, LA, and/or ALA.

4. Method according to clauses 1, 2 or 3, wherein the 20 sludge originates from fish production, sugar processing, and/or communal sludge plants.

5. Method according to any of clauses 1-4, wherein the aquatic worms are of the class of Oligochaeta.

25 6. Method according to clause 5, wherein the aquatic worms are of the species Lumbriculus variegatus.

7. Method according to any of clauses 1-6, further 30 comprising the step of separating the waste sludge, worm faeces and worms, using separating means.

25 8. Method according to any of clauses 1-7, wherein the aquatic worms are used as test organism to detect specific bioaccumulation and/or toxicity assays.

5 9. Use of the grown biomass with the method according to any of clauses 1-8 as consumption fish feed.

10. Device for growing worm biomass, comprising: a reactor for the worms; 10 - aquatic worms provided in 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.

Claims

1. Werkwijze voor het groeien van biomassa op een slib, omvattende de stappen: 5. het voorzien van waterwormen in een reactor; het voorzien van een slib voor het groeien van biomassa omvattende specifieke componenten; het aanvoeren van slib naar de reactor bevattende wormen. 10A method for growing biomass on a sludge, comprising the steps of: 5. providing water worms in a reactor; providing a sludge for growing biomass comprising specific components; supplying sludge to the reactor containing worms. 10

2. Werkwijze volgens conclusie 1, omvattende eiwitten, vetzuren en/of aminozuren.Method according to claim 1, comprising proteins, fatty acids and / or amino acids.

3. Werkwijze volgens conclusie 2, waarin de vetzuren 15 omvattende meervoudige onverzadigde omega-3 en omega-6 EPA, DHA, AA, LA, en/of ALA.3. A method according to claim 2, wherein the fatty acids comprising polyunsaturated omega-3 and omega-6 EPA, DHA, AA, LA, and / or ALA.

4. Werkwijze volgens conclusie 1, 2 of 3, waarin de slib afkomstig is van visproductie, suikerverwerking en/of 20 afvalwaterzuivering.4. Method according to claim 1, 2 or 3, wherein the sludge comes from fish production, sugar processing and / or waste water treatment.

5. Werkwijze volgens één of meer van de conclusies 1-4, waarin de waterwormen van de klasse van Oligochaeta zi jn. 25The method according to one or more of claims 1-4, wherein the water worms are of the class of Oligochaeta. 25

6. Werkwijze volgens conclusie 5, waarin de waterwormen van het soort Lumbriculus variegatus zijn.The method of claim 5, wherein the waterworms are of the species Lumbriculus variegatus.

7. Werkwijze volgens één of meer van de conclusies 1-6, 30 verder omvattende de stap van het scheiden van het afvalslib, worm faeces en wormen, met gebruikmaking van scheidingsmiddelen.7. Method according to one or more of claims 1-6, further comprising the step of separating the waste sludge, worm faeces and worms, using separation means.

8. Werkwijze volgens één of meer van de voorgaande conclusies 1-7, waarin de waterwormen worden gebruikt als testorganisme om specifieke bio-accumulatie en/of toxiciteitsgehalten te detecteren. 5Method according to one or more of the preceding claims 1-7, wherein the waterworms are used as a test organism to detect specific bio-accumulation and / or toxicity levels. 5

9. Gebruik van de biomassa verkregen met de werkwijze volgens één of meer van de conclusies 1-8 als consumptie visvoer.Use of the biomass obtained with the method according to one or more of claims 1-8 as a consumption fish feed.

10. Inrichting voor het groeien van wormen biomassa, omvattende: — een reactor voor de wormen; — waterwormen voorzien in de reactor; — toevoermiddelen voor het toevoeren van slib naar de 15 wormen; en — scheidingsmiddelen voor het scheiden van de waterwormen met een specifieke componenten aan biomassa vanuit het slib.Device for growing worms biomass, comprising: - a reactor for the worms; - water worms provided in the reactor; - supply means for supplying sludge to the worms; and - separation means for separating the water worms with a specific biomass component from the sludge.