THE PROJECT

Nowadays, 178,2 million tons of the fish world production ensure a per caput average of 20,7 kg/year; however, the demand of fish products is globally increasing (3.2% growth on year basis). Fishery is being overexploited and only aquaculture can supply the growing fish product demand. One of the limits of aquaculture is represented by feed, which requires a high percentage of Fish Meal (FM) and Fish Oil (FO). With the growing demand for fish products (and aquaculture) the market availability of FM/FO has dramatically shrunk and its price has ballooned. This has a big impact on aquaculture, especially for small local business.

A possible strategy to improve feed products is to produce the essential ingredients through a biotechnological process.
The general aim of MYSUSHI is to improve the aquaculture sector through enabling biotechnologies to transform crude glycerol, a renewable industrial by-product still having the potential of being substrate for microbial bioconversions, into the essential lipid ingredients for fish feed.

Specifically, the project has two parallel objectives:

1. Biotechnological aim – development of a sustainable and viable biotransformation process producing the key lipid ingredients for fish feed (n-3 Long Chain Fatty Acids, LCFA, Poly Unsaturated FA, PUFA and carotenoids);

2. Responsible Research Innovation (RRI) aim – transfer of the key enabling technologies to aquaculture by establishing interactive processes between innovator and social actors to allow a proper embedding of technological and scientific advances in aquaculture society. This approach is willing to promote not only this specific project, but more generally biotechnological approaches as sustainable alternative answering at the increasing need for novel sources of goods and services.

State of the art

Legend:

Long Chain Fatty Acids (LCFA)

Linoleic Acid (LA)

Arachidonic Acid (ARA)

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Fish Oil (FO)

Vegetable Oil (VO)

Microbial Oil (MO)

Aquatic resources, with a total production as 178×106 tons per year, contribute to cover a significant percentage of the world proteins demand and almost fullfill the need for the rare Long Chain Fatty Acids omega-3 (n-3 LCFAs), as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), known to be essential in human nutrition.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

Generally, fishmeal (FM) together with fish oil (FO) is incorporated in fish feeds at different percentages; however, because of the increasing FM and FO costs, these percentages are constantly substituted by the addition of cheaper plant-based ingredients (Vegetal oil, VO). As a consequence, the resulting unbalance in essential amino acids (AA), the presence of anti-nutritional factors (e.g., saponins and gossypol) and palatability problems should limit the use of plants meal in the fish diets. Moreover, vegetal oil (VO) being rich of LCFA omega-6, undesirably increase the amount of linoleic acid and arachidonic acid in fish diet, instead of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The substitution of large amounts or the total of FO with VO, beside causing poor performances in aquaculture productions, reduces the nutritional value of the fish product that will go below the expected amount of EPA + DHA to cover the 500 mg/day, as recommended by the WHOs for human nutrition. This is the main gap that the project aims to overcome by introducing microbial biomasses in fish feed formulation to match the desired feed additives panel.

Other than the biotechnological aim, MySushi project have another purpose: to transfer the key enabling technologies to aquaculture by establishing interactive processes between innovator and social actors to allow a proper embedding of technological and scientific advances in aquaculture society. This approach is called Responsible Research Innovation (RRI) and is willing to promote not only this specific project, but more generally biotechnological approaches as sustainable alternative to the increasing need for novel sources of goods and services. Right now, aquaculture market lack of an alternative formulation without FO and FM, and microbial oil is the innovation that can add omega-3 in fish feed.

Image from SOFIA 2016 http://www.fao.org/3/a-i5555e.pdf

Biomass waste

Legend:

Long Chain Fatty Acids (LCFA)

Linoleic Acid (LA)

Arachidonic Acid (ARA)

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Fish Oil (FO)

Vegetable Oil (VO)

Microbial Oil (MO)

Edible biomasses are not sustainable as substrates for biotechnological use, since their high economic, environmental and social costs are unbearable. For this reason, despite the presence of inhibitory compounds for microbial cell growth, the use of raw materials and by-products is highly desirable.

Glycerol can be considered as a by-product mainly deriving from biodiesel production. Indeed, the triglyceride trans-esterification releases glycerol at the end of the process. It is estimated that for every 100 kg of biodiesel produced, 10 to 11 kg of crude glycerol are generated. Because of the rapid growth of biodiesel industry, a large surplus of glycerol has been generated. This in turn affects the market value: even if the prices are quite volatile, glycerol can be considered as a cheap carbon source, and crude glycerol is even cheaper. An efficient valorization of crude glycerol is difficult to achieve, despite this would help significantly in reducing the costs of the overall biodiesel production. The problematic in using this industrial by-product as such is mainly related to the impurities present therein: residual methanol, sodium hydroxide, some esters and low amounts of proteins and minerals. These contaminants strongly limit crude glycerol application in chemistry or pharmaceuticals without a purification step, which is costly and energy-intensive. However, microbial ability to transform an excess of carbon source into polymeric reservoirs -up to 70% of their dry weight- is well known. For these reasons, the microbial conversion of crude glycerol into value added compounds attracts increasing interest and represents an alternative and environmentally friendly strategy.

Microorganism

Legend:

Long Chain Fatty Acids (LCFA)

Linoleic Acid (LA)

Arachidonic Acid (ARA)

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Fish Oil (FO)

Vegetable Oil (VO)

Microbial Oil (MO)

Exploitation of microorganisms (i.e., biotransformation) in beverages, food and feed production is well established since ancient times in human culture; one recent example in the field of food formulation is the use of biomasses derived from cyanobacterium Spirulina platensis used as supplement product in order to fulfill the increasing demand of non-animal proteins, as for the vegan diet.

In this context, the principal project aim is to use microorganisms, mainly oleaginous yeasts and heterotrophic microalgae, described in literature for their highly efficient accumulation of  PUFA, DHA and EPA, in order to extract omega-3 and carotenoids. The principal aspects investigated in the project is the selection of specific oleaginous microorganisms able to efficiently use crude glycerol as carbon source and the optimization of growth conditions for the production of a panel of fatty acid containing the desired compositions in terms of Long Chain n-3. The second phase consist in the formulation of fish feed pellets, containing omega-3 and carotenoids. The biotechnological oil (MO=microbial oil) produced by MYSUSHi, as substitute for Fish Oil, will allow to regain a correct amount of EPA and DHA in the fish diet and therefore to improve the nutritional value of it.

Microorganism

Nutraceutical

Legend:

Long Chain Fatty Acids (LCFA)

Linoleic Acid (LA)

Arachidonic Acid (ARA)

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Fish Oil (FO)

Vegetable Oil (VO)

Microbial Oil (MO)

Since Long Chain Fatty Acids (LCFA) as n-6 and n-3 (omega 3) are assimilated only through diet, fish must be fed with omega-3-enriched feed. n-6 LCFA, as Linoleic Acid (LA) and Arachidonic Acid (ARA), are available in several cheap Vegetable Oil, while n-3 LCFA, as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are very rare and available only in Fish Oil. Therefore this project aims to select yeasts and microalgae strains that, in optimized fermentation conditions, will produce MO respectively enriched in linolenic, carotenoids, and EPA/DHA.

Fish feed

Legend:

Long Chain Fatty Acids (LCFA)

Linoleic Acid (LA)

Arachidonic Acid (ARA)

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Fish Oil (FO)

Vegetable Oil (VO)

Microbial Oil (MO)

Due to the generalized use of Vegetable Oil instead of Fish Oil, fish diet is characterize by unbalanced in essential amino acids, the presence of anti-nutritional factors, and palatability problems. The consumption of fish fed with FO substituted by VO, lead to a poor human supply in n-3 LCFAs and the recommended 2-3 fish portions per week do not supply the needed amount of 500 mg/day. Microbial oil will be evaluate to determine its digestibility to fishes and its nutritional properties, when added at established concentration and compared to optimal as well as sub-optimal fish feed formulation. Several authors have reported the need for Essential Fatty Acids (EFA) in rainbow trout where a requirement as percent of dry diet is indicated between 1.0-0.4 for linolenic acid and n-3 LCFAs.

The MO produced in MySushi will be able to contribute to cover the recommended consumption, providing a cheap and sustainable supply of EPA+DHA in farmed fish.

Gut microbiome

Little is known about the influence of microbiome on fish physiology, but it is clear that rainbow trout has a stable “core microbiome” especially in the intestine, and this is functional to maintain the immune system and digestive efficiency. A health intestine can contribute positively to a health environment inside the tanks, increasing the environmental sustainability of the production. The effect of a MO-enriched diet in comparison to a Fish-Vegetable Oil on perturbing/riequilibrating the fish microbiota is monitored by High Throughput Sequencing (HTS) techniques.