WO2024089594A1

WO2024089594A1 - Method for rearing black soldier fly and use of microalgae therein

Info

Publication number: WO2024089594A1
Application number: PCT/IB2023/060714
Authority: WO
Inventors: Edmund Franklen ANDERSON; Kiman KAREL; Eden STEVEN; Yovita Yunita ANGKAWIJAYA; Fransiska -; Clare EVANGELINE
Original assignee: Pt Hermetia Bio Sejahtera
Priority date: 2022-10-24
Filing date: 2023-10-24
Publication date: 2024-05-02

Abstract

The present invention relates to methods for rasing black soldier fly (BSF) larvae, in particular to the use of live microalgae as an additional feed component when raising BSF larvae and to the synergistic cultivation of BSF larvae and microalgae.

Description

Method for Rearing Black Soldier Fly and Use of Microalgae therein

Field of the invention

The present invention relates to a method of raising black soldier fly larvae for use as food or feed nutrition using microalgae as feed component.

Background

Rapid population growth has dramatically increased the global food demand. However, to meet such demands is a great challenge. Generally, protein is sourced from livestock such as chicken and beef where it is becoming more challenging to increase production due to the need for huge areas of land for feed crops. The problem is further complicated by extreme fluctuation and unpredictable weather that affects the crop production as their feed which is partially fueled by the greenhouse gas emissions stemming from the rearing of these animals. An alternative protein source is fish. However, accumulating pollutants in water bodies from chemical wastes and microplastics are beginning to affect the quality and yield of fisheries. Thus, further alternative protein sources are important. Besides proteins, alternative lipid sources are also important. Lipids are generally sourced from plants and some from livestock or fish thus they also experience the aforementioned challenges.

The protein and lipid profiles of various sources are different. Both amino acids and fatty acids are classified into essential and non-essential. Essential amino acids and fatty acids are components that either cannot be synthesized or are synthesized in quantities too small to meet the body's needs. On the other hand, non-essential amino acids and fatty acids are components that are normally present and synthesized in the cells. Therefore, assuring that these essential acids are present in a given diet is important. However, some essential amino acids and fatty acids are scarce in nature. Some essential amino acids are special, in which they are required for full protein synthesis in human or animal diets. Their lack or excess presence in a food/feed source dictates the protein bioavailability of the food/feed. There are not a lot of sources that can produce methionine. Mainly it is derived from fish sources. Likewise, essential fatty acids are important for healthy metabolism. Unsaturated fatty acids are known to be beneficial when consumed. In particularthe omega-3 fatty acids are considered to be the healthiest for consumption. Omega-3 fatty acids, similar to that of methionine, are difficult to find. Mainly it is derived from fish oil.

Hence there is a need for alternative sources of protein and lipid to meet the global food/feed demand, and especially for proteins and lipid with high quality nutritional values containing methionine and/or omega-3 fatty acids. Equally important is the need to establish a circular and environmentally sustainable production process with minimal or reduced greenhouse gas emission.

Insect is one of the appealing alternative resources for production of high- quality protein, lipids, and many other value-added products. In particular, the Black Soldier Fly (BSF) rearing is advantageous. BSF rearing is compatible with vertical farming concepts and may be carried out both indoors and outdoors. Thus, raising BSF is more space efficient compared to raising livestock such as beef, chicken or fish. In addition, BSF is not a disease vector as they have a very short lifespan and do not eat as flies, but only in the larval stage. This fact distinguishes them from other types of flies and insects. Furthermore, in their larval stage, BSF is known to produce probiotics that protects themselves from harmful microbes around it. BSF larvae can even metabolize certain toxins in their gut.

BSF is typically reared using organic waste material, such as food waste, fruit waste, vegetable waste or general kitchen waste. Studies have shown that BSF larvae that are reared from organic waste materials produce protein with a complete set of amino acids including methionine. This makes BSF protein highly valuable. In terms of lipid, BSF larvae raised on organic waste material produce lipid that contains a high percentage of lauric acid, a medium-chain fatty acid that is important in biomedical and cosmeceutical products.

However, there are certain limitations despite the ability of BSF larvae raised on organic waste material reared to produce high value protein and lipid substances. Methionine is usually only found in very minimal amounts in BSF protein. Other useful fatty acids such as those of the omega-3 fatty acid types are scarce or not present in most cases. Furthermore, BSF rearing using organic wastes often face challenges in terms of excessive gas buildup in the feed substrate that results in an increased of mortality rate of the larvae, and thus lowering production yield. By-products such as ammonia and carbon dioxide may be generated by the larvae and the feed substrate in excessive amounts that require additional processing to minimize its environmental impacts.

CN111011311A discloses rearing of BSF to remediate cyanobacterial mud and removal of algal toxin to counter a blue-green algae, i.e., cyanobacteria, pollution problem and at the same time obtaining insect protein.

Similarly, CN110352913B discloses the utilization of blue algae and kitchen waste to rear BSF and to counter blue-green algae pollution.

CN110526530B also discloses a method for treating blue algae using a microorganism and feeding it to maggots. It is reported that the maggots obtained can be used as a protein source the rearing waste can be used for fertilizing purposes.

While BSF may be used to counter a cyanobacteria pollution problem and yield a protein product, there is still a need to improve the nutritional quality of the BSF and/or to improve BSF production. At the same time, it is desirable to reduce the environmental impact and/or carbon-footprint of BSF production.

Summary of the invention

These and further objects are met by a method to improve Black Soldier Fly larvae rearing and nutrition using microalgae as feed component as disclosed herein. More specifically, this invention provides a method for raising Black Soldier Fly (Hermetic/ illucens, abbreviated BSF) larvae, comprising the steps of providing BSF larvae and/or BSF eggs in a rearing vessel, supplying the rearing vessel with a feed material, and supplying the rearing vessel with microalgae.

The microalgae which serves as an additional feed component in the rearing of BSF may improve the rearing effectiveness, by providing reduced larvae mortality and/or improved yield of larvae mass, improve protein and lipid quantity, quality of the BSF larvae and at the same time minimize the environmental impacts of BSF. As will be described in greater detail below in relation to specific embodiments of the invention microalgae may be used in a circular way as feed component for BSF larvae by reducing and upcycling a gas by-product of the BSF rearing process by using it for microalgae cultivation. The terms "raising" and "rearing" BSF larvae are used interchangeably herein to refer to the process of growing BSF larvae. The term "microalgae" as used herein refers to a plurality of microalgae organisms which may or may not be from the same species of microalgae.

In some embodiments, the microalgae is a green microalgae (Chlorophyta).

In some embodiments, the microalgae is selected from the group consisting of: Chlorella sp., Parachlorella sp., Auxenochlorella sp. or a combination thereof.

Preferably the microalgae comprises Chlorella sp. Chlorella sp. has been found to provide better improvement in mortality rate compared to other microalgae species.

In some embodiments, the microalgae and feed material are mixed before being supplied to the rearing vessel and/or before the step of providing BSF larvae or BSF eggs in the rearing vessel. Mixing the microalgae and feed material increases availability of microalgae to BSF larvae, but once the larvae or eggs are added to the rearing vessel, achieving sufficient mixing may be difficult without disturbing or destroying the BSF larvae or eggs. Hence, the feeds are preferably mixed prior to being mixed with BSF larvae or eggs.

In some embodiments, the microalgae are supplied as an aqueous microalgae mixture. An aqueous microalgae mixture may be more easily incorporated into the feed material. At the same time a certain moisture level is beneficial for raising BSF larvae, such as about 50 to 80 % moisture in rearing vessel (water content expressed as a percentage of dry mass in the rearing vessel), and the aqueous microalgae mixture can be used to obtain the desired moisture content, thereby replacing or reducing the need for regular rearing water. Providing the microalgae as a water mixture may also increase the likelihood of the microalgae being alive and metabolically active, which may be advantageous in maintaining the health of the BSF larvae, which will be discussed in greater detail below. In some embodiments, the aqueous microalgae mixture comprises 0.02 to 10 % microalgae by dry weight, preferably 0.02 to 6 %, more preferably 0.04 to 5 % or 0.05 to 4 % microalgae by dry weight. This may provide a good dispersion of microalgae in the rearing vessel and/or in the feed material, and may provide a concentration of microalgae therein which provides the aforementioned advantages to the BSF rearing process and nutritional quality of the BSF larvae.

According to the invention, the microalgae comprise live microalgae. Live microalgae are metabolically active, process carbon dioxide and yield oxygen. When live microalgae are supplied to the rearing vessel, they may serve as a biofilter in the rearing vessel by processing by-products generated in the rearing vessel, such as carbon dioxide and ammonia. Carbon dioxide and ammonia are by-products that may be detrimental to the growth and health of the BSF larvae, especially if high levels are reached. Carbon dioxide and ammonia are generated by the metabolism of the BSF larvae and possibly by decomposition of the feed material. The oxygen generated by the microalgae may also be beneficial for the BSF rearing environment. In this way, the live microalgae may improve the health and/or yield of BSF-larvae. The health of the BSF larvae may be observed through the mortality rate of the larvae which is the fraction of dead BSF-larvae to the total number of BSF larvae in the rearing vessel, or in a sample therefrom, at a specific time. It may also be observed by a greater yield of BSF larvae, which is the mass of BSF larvae produced in the rearing vessel. In addition, the live microalgae may reduce or eliminate emission of the by-product gasses, or the need for further processing of the by-product gasses before emission, thereby reducing the environmental impact of the rearing process. The present inventors have found that the above benefits of live microalgae can be achieved even in dark rearing vessels which have no light source or do not allow light to enter rearing vessel. Without being bound by theory, the microalgae may have residual energy deposits allowing them to continue metabolizing for a duration long enough to improve the rearing conditions of the rearing vessel. Using live microalgae is especially advantageous when the rearing vessel is configured to provide limited ventilation, as will be described in greater detail below, as the live microalgae may counter the accumulation of waste products, such as carbon dioxide and ammonia, by metabolization thereof, which accumulation may otherwise be detrimental for larvae rearing.

In an alternative, the microalgae are supplied in a dry form. This may still improve the nutritional quality of the BSF larvae and the health of the BSF larvae (improved yield and/or reduced mortality), but the advantages to the rearing conditions and health condition is less than when using live microalgae, especially in rearing vessels with limited ventilation.

In some embodiments, wherein the feed material is an organic waste material, such as kitchen waste, fruit waste matter, vegetable waste matter, agricultural waste, food processing industry waste, dairy manure, poultry waste, or human feces. BSF larvae can be raised on organic material which is a waste left-over from other products or processes. Organic is in this context used to identify material originating from living matter. Hence, BSF rearing can be used to obtain a more valuable product from organic waste, but organic waste will typically also provide an additional challenge in that it may generate a significant amount of carbon dioxide and ammonia in the rearing vessel detrimental to the health of the BSF larvae. Hence, the combination of organic waste feed material and microalgae, in particular live microalgae, may at least reduce the detrimental effects of using organic waste material. The feed material is preferably coarse, thereby providing porous bed of material where free, non-ab- sorbed, water can be observed at the base of the rearing vessel. The free volume of water may be beneficial for microalgae metabolism.

In some embodiments, an aggregate feed amount is the sum of feed material and microalgae supplied to the rearing vessel on dry weight basis, and the microalgae is less than 30 % of the aggregate feed amount by dry weight, preferably less than 25 %. Hence the microalgae may be 1 to 30 % of the aggregate feed amount by dry weight, preferably 5 to 25 %, more preferably 10 to 25 %, even more preferably 15% to 22 % or 18 to 22 %, by dry weight. Such levels of microalgae in relation to feed material is found to be effective in achieving improvements in nutritional quality and rearing conditions. Using organic waste as a feed component may present a nutritional challenge due to the irregular nutritional content of such waste streams, and it has been found that using microalgae in combination with organic waste in the amounts referred to above, may serve to close nutritional gap which is presented by the irregular organic waste component.

In some embodiments, wherein the BSF-larvae are harvested and further processed to a protein meal, preferably after a rearing period in the range of 12 to 21 days or 13 to 21 days. A rearing period of 12 days is preferred. The rearing period is calculated from the time at which BSF eggs or larvae were added to the rearing vessel. Using this rearing period, the rearing is continued while point algae is still active, while if the rearing period is prolonged the feed material may ferment and thereby absorb water, which may be detrimental to the rearing process.

Processing the BSF larvae into a protein meal may comprise drying and grinding of the BSF larvae into a meal or flour and methods for doing so are known to the skilled practitioner.

In some embodiments, the rearing vessel comprises a base and side walls extending along a perimeter of the base, which base and side wall thereby define a rearing volume of the vessel, and the rearing vessel is covered by a lid arrangement provided with a plurality of first ventilation holes. Such a rearing vessel limits the exchange of gas with the surroundings and thus reduces evaporation from the rearing volume. These rearing vessels are referred to as rearing vessels with limited ventilation. By reducing evaporation, the risk of the rearing vessel drying out, which can lead to increased mortality rate, is reduced. However, limiting ventilation from the rearing vessel has the detrimental effect of limiting removal of waste products, such as carbon dioxide and ammonia, which is also detrimental to the rearing process. This detriment may be mitigated by the presence of live microalgae in the rearing vessel, which serves to reduce the effect of the waste products, and thus contributing the maintaining healthy rearing conditions in the rearing vessel, even with limited ventilation. The rearing volume is the volume in which the BSF larvae are raised, and is delimited by the base, side walls and the lid arrangement. The plurality of first ventilation holes are distributed in a part of the lid arrangement, which part delimits the rearing volume, so as to provide fluid communication directly from the rearing vessel through the first ventilation holes.

In a further development, the lid arrangement comprises a lid base, a lid covering and lid side walls, which lid base covers the rearing volume of the rearing vessel and comprises the plurality of first ventilation holes, which lid covering is provided above and spaced apart from the lid base to define a lid volume between the lid base and lid covering, and which lid side walls extend along the perimeter of lid volume and comprise a plurality of second ventilation holes. By providing the lid arrangement with the lid volume, ventilation is further limited, thereby contributing to preventing the rearing vessel from drying out. The first ventilation holes provide fluid communication from the rearing volume to the lid volume, while the plurality of second ventilation holes provide fluid communication from the lid volume to the surroundings of the rearing vessel. This lid arrangement may also allow several rearing vessels to be stacked on top of each while still providing some ventilation, which increases larvae production per area. The lid arrangement may comprises a plurality, such as two, side walls extending concentrically. This further limits ventilation. Concentric side walls provide an inner lid volume delimited by an inner side wall and further lid volumes delimited by two concentric side walls.

The lid arrangement may be formed by a single structure or separate elements. Hence, the lid base and lid side walls may be formed as one element, while the lid covering is formed by a further separate element. Alternatively, the lid covering and the lid side walls may be formed as one element, with the lid base separate. Still further, the lid base and lid covering may be two separate elements each with projections extending so as to form the side walls of the lid arrangement, which the lid base and lid covering are installed on the rearing vessel.

In a further development, the lid arrangement is formed at least in part by a base of a further rearing vessel stacked on the rearing vessel. For example the lid covering may be formed by the base of further rearing vessel stacked on top of the rearing vessel. This may reduce material use and/or the number of components of the rearing vessel. Similarly, the lid side walls be provided as part of the base of the further rearing vessel stacked on top. Controlling the ventilation of the rearing vessel, and thus the rearing conditions, is achieved by adjusting the area available for fluid communication through the number and size of the first and/or second ventilation holes. Good rearing conditions with live microalgae may be achieved first and/or second ventilation holes having the following characteristics. In some embodiments, each hole of the plurality of first ventilation holes has a size in the range of 0.5 to 30 mm², such as 3 to 20 mm² or 5 to 15 mm². In some embodiments, each hole of the plurality of second ventilation holes has a size in the range of 7 to 70 mm², such as 15 to 50 mm² or 20 to 40 mm². A preferred embodiment comprises first ventilation holes of the above sizes and second ventilation holes of the above size. In some embodiments, a total area of the plurality of first ventilation holes constitute 2 % or less of base area of the lid volume. The base area of the lid volume refers to the area of the part of the lid arrangement which delimits the lid volume from the rearing volume below. Hence, the base area can be the area of the lid base. The first ventilation holes may constitute 0.05 to 2 %, preferably 0.1 to 1.7 %, more preferably 0.2 to 1 %, even more preferably 0.3 to 0.6 % of the base area of the lid volume. In some embodiments, a total area of the plurality of second ventilation holes constitute 4% or less than a perimeter area of the lid volume. The second ventilation holes may constitute 0.5 to 4 %, preferably 0.7 to 3 %, more preferably 1.0 to 2.5 %, even more preferably 1.3 to 2 % of the perimeter area. The perimeter area refers to the area of the part of the lid arrangement which forms the perimeter of the lid volume. Hence, the perimeter area can be the area of the lid side walls which faces the lid volume. A preferred embodiment comprises first ventilation holes and second ventilation holes constituting the above percentages of the base area and perimeter area of the lid volume respectively. The first and second ventilation holes may be distributed across substantially the entire base area and entire perimeter area of the lid volume respectively, which allows for the lid arrangement to have portions having other functionalities such as portions configured to accommodate stacking of vessels. Substantially the entire base area or perimeter area may be 80 % or more, or 90 % or more of base area and perimeter area. The first and second ventilation holes may be distributed evenly. The lid volume of the rearing vessel is generally smaller than the rearing volume, typically less than 30 % of the rearing volume or even less than 25 % of the rearing volume. Having a small lid volume increases air flow velocity in the lid volume, creating turbulence, which prevents the BSF from exiting through the ventilation holes early in the rearing period, when their size still allows them to exit.

For rearing vessels wherein the lid arrangement having concentric side walls, the above relative size of the lid volume refers to the total of the concentric lid volumes formed. Similarly the base area of the lid volume refers to a total lid volume formed by such concentric lid volumes, as this the area of the lid arrangement which delimits to the rearing volume below.

The base and side walls of the rearing vessel are generally solid, that is without ventilation holes. Similarly, the lid covering of the lid arrangement is typically solid. The rearing vessel may configured to be stackable, which means that that a rearing vessel or the lid arrangement thereon is configured to receive the base of a further rearing vessel and support the weight of the further rearing vessel and the contents there. Plastic is a suitable material for the rearing vessel and its lid arrangement. A typical shape for the rearing vessel and its lid arrangement is rectangular, in which case there are four side walls extending along the perimeter at an angle to each other. Circular rearing vessels are also possible, in which case the side walls is typically a single circumferential side wall. The rearing vessel preferably has a height in the range of 10 to 30 cm, preferably 12 to 25 cm. By having a low bin the mass of material per unit area in the rearing volume during rearing is also kept low, preventing compaction of the material, which would be detrimental for the rearing.

The present method is concerned with large scale production of BSF larvae. In laboratory scale, maintaining optimal rearing conditions can be achieved by rearing the larvae in a climatic chamber where temperature, humidity and gas composition can be controlled. Doing the same in large scale is not feasible, so alternative measures are needed to control the rearing conditions. The method according to the invention achieves this by the use of live microalgae in the rearing vessel and in particular embodiments, in combination with rearing vessels with limited ventilation as described above. Hence, the invention is suited for large scale production of BSF larvae.

Such large scale production, may be when the rearing vessel has a rearing volume of at least 0.01 m³, and preferably wherein the method comprises raising BSF in a plurality of such rearing vessels, and more preferably with the rearing vessel arranged in one or more stack(s) of rearing vessels. The rearing vessel may have a rearing volume of at least 0.25 m³, at least 0.4 m³ or even at least 0.5 m³. The method may be carried out in a facility comprising at least 100 rearing vessels or event at least 1000 in a building.

In some embodiments, the rearing vessel is translucent allowing light to enter the rearing vessel. Although it has been found that the microalgae can metabolize harmful by-product gasses in dark vessels as previously described, allowing some light to enter the rearing vessel may increase the metabolization of harmful waste-gasses by the microalgae and further improve the rearing conditions for the BSF larvae. The translucent rearing vessel may be partially translucent, such as allowing less than 50 %, 40 %, 30 %, 20 %, but more than 10 % of light applied to the rearing vessel to enter the rearing vessel.

In some embodiments, the methods further comprise suppling the rearing vessel with a green macroalgae. The green macroalgae is preferably Spirogyra sp or selected from the genus Bryopsis. Such green macroalgae are source of protein and may improve the nutritional value of the BSF larvae, in particular the protein content. The microalgae will typically have a higher crude protein content than the green macroalgae, but it is typically more costly, and using a combination of green macroalgae and microalgae may provide a cost-efficient method for rearing BSF-larvae, which improves nutritional quality and larvae health.

Spirogyra sp is sometimes an unintended organic waste material from eutrophic water/aquaculture systems. Using it as a feed component in the present methods provides a value-adding way to utilize the waste.

Bryopsis is also known as hair-algae. It is presently contemplated that supplying the rearing vessel with Bryopsis macroalgae, may comprise incubating the feed component, which is preferably an organic waste material, with the Bryopsis macroalgae, thereby growing the Bryopsis macroalgae on the feed component. This may improve the nutritional value of the feed component. In such an embodiment, the feed component, which is preferably an organic waste material, should contain fiber, such as fruit or vegetable matter. The green macroalgae may be supplied in addition to the feed component or alternatively it may be the feed component. Hence, the rearing vessel may be supplied with microalgae, green macroalgae and the feed component. In an alternative embodiment, the feed component is green macroalgae, and the rearing vessel is supplied with green macroalgae and microalgae. In one embodiment, the vessel is supplied with organic waste material as the feed component; with Chlorella sp., Parachlorella sp., Auxenochlorella sp. or a combination thereof, as the microalgae; and with Spirogyra sp or a green macroalgae selected from the genus Bryopsis.

While not presently considered preferable, the present inventors also contemplate rearing BSF-larvae with a feed comprising the green macroalgae as described above, without the supplying the rearing vessel with microalgae. Such a method would comprise suppling the rearing vessel with a feed component and green macroalgae, where the feed component and green macroalgae are as described herein.

In some embodiments, the method further comprises cultivating microalgae in a cultivation vessel, supplying the rearing vessel with microalgae by transferring microalgae from the cultivation vessel, preferably directly from the cultivation vessel, transferring gas from the rearing vessel to the cultivation vessel, preferably directly into a cultivation liquid of the cultivation vessel. This provides a circular process where any carbon dioxide in the gas of the rearing vessel can be utilized as a feed for cultivating microalgae, and any ammonia in the gas can serve as a nitrogen source for microalgae cultivation, reducing emissions from the process and improving the climate footprint and/or environmental impact of the process, while the cultivated microalgae is used as feed component for the BSF larvae. Transferring the microalgae from the cultivation vessel to rearing vessel may also ensure that the microalgae is alive when it is added to rearing vessel. The microalgae may be transferred directly from the cultivation vessel to the feed material and mixed therein before the aggregate feed is added to the rearing vessel. The gas is preferably added directly into the cultivation liquid, i.e., sparging, so as to absorb gas into the cultivation liquid.

The cultivation vessel for the microalgae may be a photobioreactor, such as a tubular glass bioreactor.

Determining whether the microalgae added to the rearing vessel are alive may be done by observing photosynthesis or respiration activity. This may be done by detecting CO2 concentration which changes due to photosynthesis activity and respiration activity. CO2 concentration may be detected for example and IR probe or by a pH indicator such as a hydrogen carbonate indicator.

The method for raising BSF larvae as disclosed herein is typically carried out as batch process, where feed material, microalgae and BSF larvae are added to rearing vessel and harvested after a suitable period of time. Typically, the feed material and microalgae, optionally pre-mixed, are added to the rearing vessel before the BSF larvae or eggs. It may also be possible to carry out the method for BSF raising as a fed batch process, where feed material and/or microalgae are added during the rearing process.

Adding BSF larvae or BSF eggs to the rearing vessel may be a manual operation where BSF larvae or BSF eggs harvested from an egg collection process is added to rearing vessel.

In the method according to invention, supplying feed material and microalgae to the rearing vessel increases the produced mass of BSF larvae, compared to BSF larvae raised in a rearing vessel supplied only with the feed material.

In the method according to the invention, supplying feed material and live microalgae to the rearing vessel reduces the mortality rate of the BSF larvae, compared to BSF larvae raised in a rearing vessel supplied only with the feed material. Mortality rate is the fraction of dead larvae in the rearing vessel or in a sample therefrom.

In the method according to the invention, supplying feed material and microalgae to the rearing vessel increases the protein content of the BSF larvae, compared to BSF larvae raised in a rearing vessel supplied only with the feed material. Protein content is here measured by any suitable method for measuring protein content such as the method according to SNI 01-2891-1992.

In the method according to the invention, supplying feed material and microalgae to the rearing vessel increases the content of essential amino acids, such as methionine, isoleucine and valine, of the BSF larvae, compared to BSF larvae raised in a rearing vessel supplied only with the feed material. The amino acid profile or content of specific amino acids may be evaluated by any suitable method for doing so, such as the method according to Acquity UPLC H-Class and H-Class bio amino acid analysis by Waters (2012). L-tryptophan may be evaluated using a method in AOAC 988.15 (2005).

In the method according to invention, supplying feed material and microalgae to the rearing vessel increases the content of omega-3, omega-6 and/or omega-9 fatty acid of the BSF larvae, compared to BSF larvae raised in a rearing vessel supplied only with the feed material. The content of omega-3, omega-6 and/or omega-9 fatty acid may be evaluated by any suitable method for doing so, such as the method according to AOCS Ce 2-66 (1993).

When comparing BSF larvae raised with microalgae and BSF larvae raised without microalgae for the purposes described above, the other conditions in the rearing process are kept the same.

In further aspect of the invention, the invention provides use of microalgae as a feed component in raising Black Soldier Fly (Hermetia illucens, abbreviated BSF) to increase mass of BSF produced, increase protein content of the BSF, lower mortality rate of the BSF, increase the content of essential amino acids, such as methionine, isoleucine and valine, of the BSF and/or to increase the content of omega-3, omega- 6 and/or omega-9 fatty acid of the BSF.

In a still further aspect of the invention, there is provided a protein meal precursor consisting of Black Soldier Fly (Hermetia illucens, abbreviated BSF) larvae pulp, containing at least 35 % protein by dry weight or at least 40 % protein by dry weight, such as 35 to 45 % protein by dry weight. A pulp of BSF larvae as used herein is understood to be a mass of crushed BSF larvae, which may optionally be dried. These and other features of the present invention will be better understood from the following detailed description of the invention.

Throughout this document, the terms "illustrates", "discloses", "demonstrates", "displays", "shows", "comprising" or "comprises" do not exclude other possible steps, process, and/or elements. Additionally, the use of "a" or "an" herein in the disclosed invention should not be interpreted as excluding any plurality.

Brief Description the Drawings

In the following embodiments the invention will be described with reference the exemplary drawings, in which

Fig. 1 is a schematic drawing of a rearing vessel provided with a lid arrangement for rearing BSF larvae.

Detailed Description of the Invention

Fig. 1 shows stackable two rearing vessels 1, 2 for rearing BSF larvae (not shown) in an exploded view with rearing vessel 2 above rearing vessel 1 with a lid arrangement 3 in between. The rearing vessel 2 is identical to the rearing vessel 1 and they will be described collectively. The lid arrangement 3 covers rearing vessel 1 to close off a rearing volume 10 which is defined by side walls 11 and base 12 of the rearing vessel 1. The base 12 is rectangular and the four the side walls 11 extend along a perimeter of the base 12. The lid arrangement 3 has a plurality a first ventilation holes 31. The first ventilation holes 31 are provided in a lid base 30 of the lid arrangement 3 which lid base 30 is the part covering the rearing volume 10. The lid base 30 further has a groove 32 adapted to receive the base 12 of the rearing vessel 2, whereby the rearing vessels 1 and 2 are stackable. The lid arrangement 3 further comprises lid side walls 33 which in this embodiment is provided as part of the base 12 of the rearing vessel. Similarly, the lid arrangement 3 also comprises a lid covering which in this embodiment is provided by the base 12 of the rearing vessel 2. Lid side walls 33 are provided, which in the embodiment shown is two sets of lid side walls 331, 332 extending concentrically along a perimeter of the base 12 of the rearing vessel 2. The lid side walls 33 are provided with a plurality of second ventilation holes 330. The lid covering, provided as base 12, is seen to be solid, i.e. without ventilation holes, whereby the lid base 30, base 12, and lid side walls 33 define a lid volume when the rearing vessels are stacked (not shown), which lid volume (now shown) is ventilated by the first ventilation holes 31 and second ventilation holes 330. The lid volume (not shown) provided by the lid base 30, base 12 of rearing vessel 2 and the side walls 33 is in this embodiment provided as two concentric lid volumes separated by inner lid side walls 332. The inner lid side walls 332 thus delimit an inner lid volume and outer side walls 331 delimit an outer lid volume, the two of which form the lid volume (may be referred to as total lid volume). The lid side walls may alternatively be provided part of the lid base 30, forming a raised rim thereof (not shown).

In the embodiment shown, each of the first ventilation holes 31 has as size of about 7 mm². The size of each of the second ventilation holes 330 is about 30 mm². The total area of the first ventilation holes 31 affect the evaporation and ventilation from the rearing volume 10. In this embodiment, the total area of the first ventilation holes 31 is about 0.4% of a base area of the lid volume. The base area of the lid volume is the area of the lid base 30 which delimits the lid volume from the rearing volume 10 below, where lid volume here refer to the total lid volume formed by the concentric inner and outer lid volumes described above. The total area of the second ventilation holes is about 1.7 % of a perimeter area of the lid volume with which the ventilation holes provide fluid communication. The perimeter area is formed by the part of the lid arrangement 3 which delimits the perimeter of the lid volume with which the second ventilation holes communicate. In the embodiment shown, the total area of second ventilation holes 330 formed in the inner lid side walls 332 is about 1.7 % of the perimeter area formed by inner lid side walls 332. Similarly, the second ventilation holes 330 formed in the outer lid side wall 331 is about 1.7 % of the perimeter area formed by outer lid side walls 331.

The rearing vessel 1 may have rearing volume 10 of at least 0.01 m³ and even at least 0.05 m³. Vessels with smaller volume may preferably be used as a nursery vessel, i.e. for hatching BSF eggs, whereas larger volumes are preferred for the subsequent rearing of the BSF larvae. For production scale rearing, the rearing vessels can be stacked in stacks of for example 5-10 on top of each other, and a plurality of such stacks can be provided in a chamber of building (typically a warehouse), whereby the number of rearing vessels in the chamber can number at least 100 and even at least 1000. Obtaining good rearing condition in each rearing bin under such production scale conditions is a challenge, as the climatic system of the building cannot generally provide adequate control of the individual rearing vessel, nor is it economically viable to provide separate climatic control system for each rearing vessel. By providing the closed rearing vessel design embodied by Fig. 1 and using live microalgae, the rearing conditions of the rearing vessels may be improved in an efficient and effective manner for large scale production.

The rearing vessels 1,2 is preferably shallow, i.e. the height of the side walls 11 is small, and in the embodiment shown the height of side walls 11 is about 15 mm.

The microalgae utilized in this invention is live microalgae, such as a microalgae water mixture that is added into various desired feed materials, such as those based on organic food wastes, empty fruit bunch, and other feed material types. Empty fruit bunch has low water absorption ability due to its lignin content, which is beneficial for providing free water as previously described.

The moisture content in the rearing vessel should be maintained at a level of 50% to 80% of the mass of dry rearing media. This can be controlled by adding water and/or microalgae and/or microalgae water. Maintaining the moisture content is also facilitated by reducing evaporation by using a rearing vessel with limited ventilation as described above.

As previously described the addition of microalgae water in the present invention may reduce the accumulation of ammonia and carbon dioxide in the rearing vessel which leads to the improvement of the overall health of the larvae. This can be observed by an improved yield of the process. A reduction of bad odor from the rearing vessel can also be observed. Bad odor in this context is a sour and/or ammonia- like smell when microalgae are not present, which is replaced by a sweetersmell when using microalgae as part of the feed. The present invention and the advantages thereof are now illustrated by way of the following non-limiting examples.

Example 1

This example demonstrates advantages of using microalgae powder supplementation by comparing it to a control feed based on organic food waste. Rearing was done in the same conditions with controlled amounts of BSF egg, feed and moisture level.

330 g of feed (dry weight) with a moisture content of 75% was used in each rearing vessel and 1.2 grams of BSF eggs and the larvae were reared for 13 days before harvest. In the control rearing vessel, the feed was 330 g (dry) of kitchen waste. In the other rearing vessel, the feed was 330 g (dry) of microalgae powder. The microalgae species used in this example was Chlorella vulgaris.

Rearing using microalgae supplementation feed produced 533.06 g of fresh larvae, which corresponds to 143.93 g of larvae on a dry weight basis. The obtained yields are shown in Table I. In the control vessel, rearing using the organic food waste produced 239.94 g of fresh larvae, which corresponds to 64.78 g of larvae on a dry weight basis. This corresponds, on a dry-to-dry weight basis, to a feed conversion ratio feed:larvae of 2.3:1 for the larvae fed with microalgae supplementation, whereas a less efficient feed conversion ratio feed:larvae of 5.1:1 was observed with the larvae fed only on organic food waste. Hence, using microalgae in addition to organic waste increased the yield of produced larvae.

Table I

Example 2

This example shows the crude protein, lipid, mineral and carbohydrate contents of BSF larvae of example 1 reared with microalgae supplementation compared to those reared only on organic food waste feed. Also included are the compo- sition of the microalgae and organic food waste feed.

Crude protein was measured using the methods disclosed in SNI 01-2891-

1992. Carbohydrate was measured through by-difference method, i.e., carbohydrate constitute the part which is not protein, fat, moisture or minerals.

The results are shown in Table II, which shows that BSF larvae fed with mi- croalgae supplementation had an improved crude protein content at 62.33% per dry weight compared to 40.61% with control organic food waste. The fat content was also reduced in the BSF larvae which were fed microalgae.

Table II

Example 3

In this example, the synergistic interaction between the BSF larvae and microalgae supplementation from example 1 is shown, which results in an improved amino acid profile. The amino acid content was measured through a method based on acquity UPLC H-Class and H-Class bio amino acid analysis by Waters (2012). L-tryp- tophan was measured through a method based in AOAC 988.15 (2005). Shown in the table III below are the amino acid profiles of the larvae fed with microalgae supplementation compared to those fed only on organic food waste feed. BSF larvae fed with microalgae supplementation had an improved amount of total amino acid content at 60.79% (per whole larvae dry weight basis) compared to the 37.88% (per whole larvae dry weight basis) found in BSF larvae fed only on the organic food waste. An improved production of methionine is especially observed for BSF larvae fed with microalgae reaching the values of 14.80% (per whole larvae dry weight basis) com- pared to those fed with organic food waste with a much lower value of 0.38% (per whole larvae dry weight basis). This is especially surprising in light of the low to moderate percentage of methionine found in the microalgae feed itself, 0.33%, which highlights that the improved production of methionine in the BSF larvae is a synergistic interaction between the BSF larvae and microalgae feed. Other improvements of BSF larvae fed with microalgae supplementation could be observed in isoleucine and valine content at 2.09% and 3.10%, respectively; compared to the content within BSF larvae fed with organic food waste feed at 1.18% and 1.73%, respectively. In general, the BSF larvae raised with microalgae had a larger content of essential amino acids, which are especially important for protein food/feed and increase the nutritional value thereof.

Table III

Example 4

This example shows the Omega 3, Omega 6, and Omega 9 fatty acid content of BSF larvae fed with microalgae supplementation compared to BSF larvae fed only on organic kitchen waste. The BSF in this example were raised as described in example 1. For further comparison, the fatty acid content of BSF larvae raised on three different amounts of microalgae was measured: Single quantity as done in example 1, twofold quantity and five-fold quantity.)

Fatty acid contents were measured through a method mentioned in AOCS Ce 2-66 (1993).

An increasing trend of Omega 3 in BSF larvae fed with increasing quantity of microalgae supplementation can be observed, with 0 % at single quantity, 0.735 % (9.09% of total lipid) at two-fold quantity and 1.552% (19.47% of total lipid) at fivefold quantity. In addition, ratio of each omega fatty acid to total lipid in BSF larvae fed with microalgae supplementation show a significant improvement compared to BSF larvae fed with organic food waste. Omega 3 and Omega 6 fatty acids in BSF larvae fed with microalgae supplementation show an improvement at 9.09% and 16.69% of total lipid content (twice the microalgae supplementation quantity), respectively; compared to BSF larvae fed with organic food waste at 2.58% and 5.34% of total lipid, respectively. Even with a single quantity, Omega 6 fatty acid in BSF larvae fed with microalgae supplementation still shows an improved quantity at 13.80% of total lipid.

5 Example 5

This example demonstrates the rearing of black soldier fly in bins with limited ventilation and therefore wet environment, compared to an open bin with a dry environment. Microalgae is not used in these examples.

The "Wet bin" is a rearing vessel having the lid arrangement. The "Dry bin" 0 is a rearing vessel without the lid arrangement. As such, moisture content in the dry bin must be closely regulated to compensate for evaporation. Therefore, BSF larval egg was hatched in nursery bin prior to introduction to the dry bin, or they would risk dehydration. The BSF larvae spent upto 6 days in nursery bin with volume of 29x23x16 cm³, rearing temperature of 31-35 °C and ambient humidity of 50-70%. A total of 0.5 5 grams of BSF larval egg was grown in the nursery bin for 6 days before being transferred to dry rearing bin at day 7. In the dry bin, 1.5 kilograms of coarse empty fruit bunch substrate with 70% of moisture content was spread across the rearing vessel which has volume of 77x52x16 cm³. Then, 2-2.5 kilograms of a formulated feed was added on top once every 2 days during rearing process. The total feed needed up until harvest amounted approximately 8.5 kilograms. The BSF larvae previously grown in the nursery bin was then introduced to the bin. The BSF larvae spent between 7 to 8 days before being harvested. The rearing conditions included average ambient temperature of 35 - 37 °C, ambient humidity of 50-70% and substrate moisture of 70% for optimum rearing of BSF larvae. However, the temperature could elevate up to 43 °C during feeding activities.

On harvest (day 13), the BSF larvae count was 10,000-12,000 with a total weight of approximately 700-1,200 grams. This translates to approximately 50- 70 mg per larva, bin. Mortality rate for the dry bin was estimated to be around 25%.

The wet bin was a rearing vessel similar to the one in Fig. 1 with volume of 82x45x15 cm³. The wet bin was filled with 7 kilograms of the same formulated feed along with 1.5 kilograms of coarse emptyfruit bunch substrate. 0.5 grams of BSF larval eggs was placed in a box mesh and suspended on the formulated feed. The rearing bin is then left undisturbed for 14 days. The bin was closed tight with a lid arrangement. Forced air ventilation systems were put next to the bin to ensure turbulent air flow and promote an exchange of gasses, internal to the bin with external fresh air, ensuring the correct environment within the bin and promoting the required BSF larvae growth rate. The rearing condition included ambient temperature of 35 - 37 °C, ambient humidity of 50-70% and substrate moisture of 80% for optimum rearing of BSF larvae. Larval egg was incubated with minimal presence of light to curtail any stresses for the larvae.

On harvest (day 13), BSF larvae count was 12,711 with a total weight of 1,932 kilograms. This translates to approximately 152 mg per larva. Mortality rate for this method was estimated to be less than 5%.

As can be seen, the wet bin performed better than the dry bin in terms of mortality rate and larvae size even when steps were taken to regulate the moisture content of the dry bin by continually adding fresh feed to the dry bin during the rearing period. Hence, the rearing vessel with the lid arrangement provides a simple and effective manner to control the rearing process and conditions. Had the moisture content of the dry bin not been controlled by continuously adding fresh feed, it is speculated that the performance of the dry bin had been even poorer in terms of mortality rate and larvae size, than in the present example.

Example 6

This example demonstrates the advantage of using ch lorella microalgae during BSF rearing process by comparing larval survivability rate after rearing process in the presence or absence of ch Io re Ila microalgae.

Larvae used in this example primarily came from 0.75g BSF eggs reared in a nursery bin (29x23x16cm³) with ambient humidity of 50-70% at 31-35 °C. 563g feed material was adjusted to acquire a moisture content of 70-80%. BSF larvae spent 6 days in the nursery bin after which they were transferred to the rearing vessels.

The rearing vessels (82x45x15 cm³) were similar to the one in Fig. 1, and were prepared by first layering empty fruit bunch coarse fibres as base materials. The respective feed materials for the two vessels had been mixed with chlorella water and reverse osmosis (RO) water respectively and were then introduced into the rearing bin. The Chlorella water had a concentration of 0.2% and contained live Chlorella (supplied directly from its cultivation vessel). The feed materials were organic feeds with 26.71% crude protein content, 25.23% lipid content, 9.10% mineral content, and 38.96% carbohydrate content. The composition was 10.87 % feed materials 10.43% base material, 78.7 % chlorella microalgae water or RO water, with total weight of 2510 g.

At this moisture level and placement of the empty fruit bunch at the base of the rearing vessel, free algae water was seen at the bottom of the bin. This may be beneficial for promoting microalgae heterotrophic growth to scavenge dissolved ammonia or nitrate.

On day six larvae were then introduced into the rearing vessels. The rearing bin was then fitted with its lid arrangement and left undisturbed for 7 days, after which larvae were harvested.

BSF rearing in the presence of chlorella microalgae produced 3298g larvae with individual larval sizes of 136 mg. BSF larvae survivability rate was at 81% with mortality rate of 19%. Contrarily, BSF rearing in with RO water in the absence of microalgae, produced 2554g larvae with individual larval sizes of 122 mg. BSF larvae survivability rate was at 70% with mortality rate of 30%.

Survival rate was measured with a reference that 100% hatching rate will yield 40,000 larvae from 1 gram of BSF egg. The survival rate was the total individual larvae harvested divided by the reference number.

This example shows that chlorella microalgae can improve the survivability of BSF larvae during rearing process. An improvement of 11 percentage points in mortality rate was achieved. It is speculated that this is at least in part due to the ability of the microalgae to suppress larval suffocation and promote external feedback inhibition of BSF larvae.

The achieved mortality rates and larvae sizes in Examples 5 and 6 may not be directly comparable, as the feed material and other rearing conditions were not necessarily controlled to be similar.

Example 7

This example demonstrates improvement in microalgae density cultivated with using the gas from the surroundings of the rearing vessels ("rearing gas") compared to using atmospheric gas.

Microalgae cultivation was conducted in horizontal photobioreactor (Vari- con Aqua Solutions) for 7-14 days at 25-30 °C. The air source (rearing gas/atmospheric gas) for the photobioreactor was supplemented with air pump and distributed into the system using water flow pump.

The supplemented rearing gas may contain higher amount of ammonia when compared with atmospheric gas mainly due to the larvae producing ammonia during rearing process. The ammonia present in the respective gasses were converted into nitrate by bacteria in the microalgae cultivation system. The nitrate residue supplied during microalgae cultivation provides available nutrient for microalgae to grow. Additionally, phosphate residue should be present at a certain level to further supplement the growth of microalgae. Following 5-6 days of microalgae cultivation period, both nitrate and phosphate residues were analysed using Salifert nitrate test kit and Salifert phosphate test kit. Microalgae supplemented with rearing gas contained a total nitrate residue of 50 ppm with phosphate residue of >3 ppm. Contra rily, the microalgae supplemented with atmospheric gas contained a total nitrate residue of 0 ppm with phosphate residue of >3 ppm. Additionally, microalgae cultivated with rearing gas exhibited 0.672 g/L density, while microalgae cultivated with atmospheric gas exhibited 0.492 g/L, insinuating that microalgae flourished better when supplied with rearing gas due to the higher presence of ammonia in the gas.

Example 8

This example provides a comparison of mortality rates of rearing without microalgae to a rearing using both chlorella and spirulina microalgae, and to a rearing using only chlorella microalgae.

For each of the three rearing tests the mortality rate was collected for batches across four-month periods. Separate data was collected for both production batches having a 12-day rearing period and supporting batches used to produce new eggs, which had a 23-day rearing period. Rearing vessels with limited ventilation were used in all batches, i.e. vessels similar to the one schematically shown in Fig. 1, and the microalgae were supplied directly from their cultivation vessel and were thus alive.

For the batches without algae, the production batches had a mortality rate of 27.4 % in total across the four-month period. The supporting batches had a mortality rate of 22.6 % in total across the four-month period.

For the batches with chlorella and spirulina, the production batches had a mortality rate of 0.6 % in total across the four-month period. The supporting batches had a mortality rate of 8.9 % in total across the four-month period.

For the batches with chlorella only, the production batches had a mortality rate of 0.0 % in total across the four-month period. The supporting batches had a mortality rate of 0.2% in total across the four-month period. Adding microalgae to the rearing thus lowered the mortality rates in both production and supporting batches, and using chlorella alone provided better improvement than the mixture of spirulina and chlorella.

Claims

C L A I M S

1. A method for raising Black Soldier Fly (Hermetic/ illucens, abbreviated BSF) larvae, comprising the steps of:

- providing BSF larvae and/or BSF eggs in a rearing vessel,

- supplying the rearing vessel with a feed material, and

- supplying the rearing vessel with live microalgae.

2. The method according to claim 1, wherein the microalgae are selected from the group consisting of: Chlorella sp., Parachlorella sp., Auxenochlorella sp. or a combination thereof.

3. The method according to claim 2, wherein the microalgae comprise Chlorella sp.

4. The method according to any one of the preceding claims, wherein the rearing vessel comprises a base and side walls extending along a perimeter of the base, which base and side wall thereby define a rearing volume of the vessel, and the rearing vessel is covered by a lid arrangement provided with a plurality of first ventilation holes.

5. The method according to claim 4, wherein the lid arrangement comprises a lid base, a lid covering and lid side walls, which lid base covers the rearing volume of the rearing vessel and comprises the plurality of first ventilation holes, which lid covering is provided above and spaced apart from the lid base to define a lid volume between the lid base and lid covering, and which lid side walls extend along the perimeter of lid volume and comprise a plurality of second ventilation holes.

6. The method according to claim 4 or 5, wherein the lid arrangement is formed at least in part by a base of a further rearing vessel stacked on the rearing vessel.

7. The method according to claim anyone of claims 4 to 6, wherein each hole of the plurality of first ventilation holes has a size in the range of 0.5 to 30 mm², and/or wherein each hole of the plurality of second ventilation holes has a size in the range of 7 to 70 mm².

8. The method according to claim any one of claims 4 to 7, wherein a total area of the plurality of first ventilation holes constitutes 2 % or less of a base area of the lid volume, and/or wherein a total area of the plurality of second ventilation holes constitute 4 % or less of a perimeter area of the lid volume.

9. The method according to claim any one of the preceding claims, wherein the rearing vessel has a rearing volume of at least 0.01 m³ or at least 0.5 m³, preferably wherein the method comprises raising BSF in a plurality of rearing vessels, preferably arranged in one or more stack(s) of rearing vessels.

10. A method according to any one of the preceding claims, wherein the microalgae and feed material are mixed before being supplied to the rearing vessel and/or before the step of providing BSF larvae and/or BSF eggs in the rearing vessel.

11. The method according to any one of the preceding claims, wherein the microalgae are supplied as an aqueous microalgae mixture.

12. The method according to claim 11, wherein the aqueous microalgae mixture comprises 0.02 to 10 % microalgae by dry weight, preferably 0.02 to 6 %, more preferably 0.04 to 5 % or 0.05 to 4 % microalgae by dry weight.

13. The method according to any one the preceding claims, wherein the feed material is an organic waste material, such as food waste, fruit matter or vegetable matter, agricultural waste, food processing industry waste, dairy manure, poultry waste, or human feces.

14. The method according to any one the preceding claims, wherein an aggregate feed amount is the sum of feed material and microalgae supplied to the rearing vessel on dry weight basis, and the microalgae is less than 30 % of the aggregate feed amount by dry weight, preferably less than 25 %.

15. The method according to any one the preceding claims, wherein the BSF- larvae are harvested and further processed to a protein meal, preferably after a rearing period in the range of 13 to 21 days.

16. The method according to any one the preceding claims, wherein the rearing vessel is translucent allowing light to enter the rearing vessel.

17. The method according to any one of the preceding claims, further comprising

- cultivating microalgae in a cultivation vessel,

- supplying the rearing vessel with microalgae by transferring live microalgae from the cultivation vessel, preferably directly from the cultivation vessel,

- transferring gas from the rearing vessel to the cultivation vessel, preferably directly into a cultivation liquid of the cultivation vessel.

18. Use of live microalgae as a feed component in raising Black Soldier Fly (Hermetic! illucens, abbreviated BSF) to lower mortality rate of the BSF.