Valorisation of waste streams from by-product to worm biomass

There is a global demand for more feed resources to keep up with the increasing production of livestock. The hunger for resources is most urgent in the aquaculture sector, which to a large degree depends on the non-sustainable use of fish oil/ meal from wild fish. Aquatic macro invertebrates such as the freshwater worm Lumbriculus<em> variegatus</em> (Oligochaeta, Lumbriculidae, common name blackworms, further abbreviated as Lv) are rich in proteins, lipids, vitamins and minerals. When cultivated on safe and low-grade organic wastes they can provide a sustainable fishmeal alternative for most freshwater and marine fish. <strong>Chapter 1</strong> introduces the concept of aquatic worm production on waste streams. Worm biomass composition and relevant research lines are explained. Organic waste sludges from food industries are a rich source of bio-molecules and can be upgraded to (fish) feed when fed to aquatic worms. For valorisation of waste streams by aquatic worms, as proposed in this thesis, these streams preferably are free from contaminants such as organic micro pollutants, heavy metals and pathogens. For example, this would not be the case when sewage (municipal) sludge is used as a substrate for the worms. However, such contaminated sludges may still be applied for non-food applications. Thus, the quality of the waste stream that is used as a substrate for the worms determines the application potential of the worm biomass as well as the options for downstream processing and refinery. Previous research showed that Lv can be used for reduction and compaction of sewage sludge. The consumption of (suspended) sludge particles results in a dry matter reduction of 25 - 50 % and in worm faeces that are 60 % more compact than the original waste sludge. This contributes to a significant reduction in sludge processing costs. Sludge reduction by aquatic worms is mainly studied by research groups in The Netherlands and in China. Unfortunately, it is generally accepted free swimming worms in full-scale wastewater treatment plants is extremely difficult, mainly because of large (seasonal) population fluctuations. A controlled reactor concept applying the sessile (crawling, sediment dwelling) species Lv already was developed in earlier research. The key characteristic of this reactor is a carrier material for the worms, which also functions as a separation layer between the waste stream (worm food) and a water phase used for aeration, worm harvesting and worm faeces collection. This concept also was the starting point for the development of the improved reactor concept that is described in this thesis. The two main objectives of this thesis were: (1) to assess the potential of organic waste streams and by-products for Lv production for fish feed and (2) to develop a (cost and resource) effective bioreactor for this purpose. In <strong>Chapter 2</strong> a new, standardized method is described and tested that can be used for a quantitative and qualitative assessment of the effect of different substrates on worm growth. This method not only can be used to select waste streams suitable for worm production, but also is proposed as a tool is ecotoxicology studies. The test method consists of beaker experiments with a combination of agar and sand to optimize food uptake by and growth of the worms. The effects of agar gel, sand, and food quantity were studied and evaluated for different food sources. Agar gel addition ameliorated growth conditions by reducing microbial food hydrolysis and by improving the sediment structure. This guaranteed that substrate ingestion and worm growth in the first place were the result of the food quality and the effect of other (environmental) factors was reduced. A final test with secondary potato starch sludge demonstrated the test method is appropriate for the evaluation of solid and suspended organic feedstuffs/waste streams. In <strong>Chapter 3</strong> the standardized method of chapter 2 was used for worm growth studies, focussing on the effect of carbon to nitrogen (C/N) ratios of diets on worm growth and reproduction. Growth and reproduction of Lv on different combinations of wheat based derivatives like gluten and gray starch was studied at fixed isoenergetic levels (expressed as chemical oxygen demand (COD) of the food), but at different C/N ratios. Growth and reproduction rates were compared to those on Tetramin, a substrate known to result in excellent worm growth. Growth was mainly controlled by the C/N ratio of the single and mixed wheat fraction diets. Lower C/N ratios of around 6-7 gave a much better performance than high C/N ratios of around 20. This probably was caused by Lv relying on the presence of proteins as carbon and energy source. Although growth and reproduction rates were not as high as on the control diet, the results were promising for development of a worm biomass production reactor, operating on by-products from wheat processing industries. In <strong>Chapter 4</strong> a new reactor concept for Lv cultivation on waste streams was developed and tested. In a vertical tubular reactor a centralized food compartment was surrounded by a gravel layer that mimicked the natural habitat of Lv. Secondary (biological) sludge from a potato starch processing industry was used as a clean and low value food source. The results with respect to worm growth rate, density and production and nutrient recovery were compared to the previous reactor design. Much higher worm densities were achieved (6.0 compared to 1.1 kg ww m-2 carrier material) as well as much faster Lv growth rates (4.4 - 12 compared to 1.2 % d-1). As a result the areal worm production rate was no less than 40 times higher (560 compared to 14 g ww m-2 d-1). The higher worm density, which was found to be independent of gravel size in a range of 2.4 to 8.0 mm, allowed for a significantly shorter food retention time in the reactor (~ 2.2 days compared to > 10 days for the previous reactor design). This restricted microbial mineralization of the food, making high nutrient recoveries from waste to worm biomass possible: 16-30 % COD, 19-22 % N and 9-11 % P. The high biomass density also limited the release of ammonium, which at large concentrations is toxic for the worms. However, even shorter food retention times (e.g. higher loading rates) are not recommended as a minimum microbial activity is needed for conversion of the original substrate into compounds that can be taken up by the worms. In <strong>Chapter 5</strong> worm growth, reproduction and biomass quality were evaluated on several waste streams and by-products of bacterial, animal and plant origin. The effect of 26 different diets, all applied at high food levels, on Lv growth, reproduction and fatty acid (FA) content and profile were investigated. For this purpose the standardized test method of Chapter 2 was used. In addition, it was discussed which diet composition and food sources would be most suitable for large scale production of Lv. Diets consisting of single cell biomass from bacterial or plant origin with a high protein content (C/N ratio < 8.8), high P content (C/P < 50) and low in total ammonia nitrogen (TAN) (< 20 g N/kg) gave the highest growth rates and vital worms without signs of mortality. Besides the C/P ratio of the diet, worm conditions related with the difference between test and pre-culture conditions. The starting weight of the worms seemed to have an effect on the total fatty acid content of the worms. The growth potential of a diet rich in proteins and P depends on how much TAN is associated with the diet. By blending different food sources these factors to a certain extent can be manipulated. Lv seemed to have a distinct and very stable FA composition, irrespective of the diet’s FA composition. The worms were rich in poly unsaturated FAs (PUFAs), including several w3 and w6 FAs, and contained relatively high levels of C18 and C20 PUFAs. This makes them suitable as fish feed, in particular for freshwater fish. In order to serve aquaculture feed markets with an attractive alternative to fish meal, such as aquatic worm biomass, a continuous and secure bulk production needs to be realized. In <strong>Chapter 6 </strong>the performance parameters established in chapter 4 (worm growth rate, density and biomass production rate) were used as the input for a feasibility assessment of large scale worm production on secondary sludge from the potato industry. In addition, in chapter 6 future value chains and lines of research were discussed. A hypothetical worm production system treating the surplus secondary sludge from a potato processing factory can reduce excess sludge production by 50 % in solids and 62 % in volume. This is accompanied by a daily production of 1.6 metric ton of fresh worm biomass. With a very conservative estimation of the worm density of 1.6 kg ww/m2 carrier material a footprint of the system of 217 m2 can be realized, which is at least two times smaller than with a previous reactor design without a gravel layer. With reduced sludge processing costs and a conservative market price of 1.4 €/kg dry worm biomass, worm production can already be realized at an annual rate of return of 3 years. However, the costs are highly sensitive for worm biomass stocking, reactor construction and operation. A more accurate economic assessment should be based on the results of pilot-scale research. Two general product types, whole biomass (as fish feed) and refined products can be distinguished and applied in two application areas (feed and non-food), depending on the quality of the organic (waste) sludge that the worms have been produced from. Valorisation for potential bulk markets needs further refinery of crude worm biomass into a lipid (worm oil) and a protein fraction (protein isolate). This can result in several new and unique business models in aquaculture, feed, chemical and agriculture sectors. Obviously, an assessment of economical and legislative boundary conditions needs to be part of such business models. Worm biomass is a potential high quality fishmeal replacer, with a similar or even better potential than other waste based alternatives such as single cell biomass and insects. A comparison between Lv and fishmeal with respect to crude composition, essential amino acids and FAs learns that Lv is a highly suitable fish feed source. It can provide essential amino acids at sufficiently high levels. Based on its FA composition and (relatively low) fat content, Lv can best be considered a protein source. Still, worm biomass is rich in PUFA, which could be a potential high value product for feed applications. Compared to black soldier fly and bacterial production systems, Lv shows intermediate production efficiencies, while biomass harvesting and processing probably is more easy. Additional advantages of Lv worm biomass to replace fishmeal are: 1) Lv acts as a strong natural fish attractant, 2) the growth efficiency of fish on worms is high in comparison to regular feeds, 3) the nutritional profile of worms matches that of fishmeal, 4) the worms are a natural feed source for freshwater fish and 5) the worms allow a secure and stable feed production that is independent of natural resources. Further recommendations for future research as outlined and discussed in chapter 6 are mostly related to the technical upscaling of the reactor technology and obtaining more detailed insight in controlled worm growth in response to food characteristics, reactor design and operational conditions.