PRESTON NIGEL PHILIP (AU)
SMITH DAVID MORGAN (AU)
PRESTON NIGEL PHILIP (AU)
| WO2004080196A2 | 2004-09-23 |
| US4958460A | 1990-09-25 | |||
| US20040009160A1 | 2004-01-15 | |||
| US20080135474A1 | 2008-06-12 |
The claims defining the invention are as follows:
1. A process for producing a microbial biomass comprising: a) providing a mixed population of microorganisms comprising micro algae and bacteria; b) adding a carbon source to the mixed population of microorganisms; c) adding a nitrogen source to the mixed population of microorganisms; d) culturing the mixed population of microorganisms, the carbon source and the nitrogen source to form the microbial biomass; and e) harvesting the microbial biomass.
2. A process according to claim 1, wherein the step d) of culturing is conducted in an open vessel.
3. A process according to claim 1 or 2, further comprising a step of drying the harvested biomass to form a feed product.
4. A process according to claim 2, wherein the step d) of culturing occurs in tanks, raceways or ponds.
5. A process according to any one of the preceding claims, wherein the carbon source is selected from the group consisting of products, by-products or waste streams from the processing of sugar cane, molasses, rice, wheat, triticale, corn, sorghum, tapioca, oilseeds, canola meal, lupin hulls, elevator dust from grain handling facilities, production waste from feed mills, and distillery spent grain products.
6. A process according to claim 5, wherein the carbon source is a material which has been milled or sieved.
7. A process according to claim 5 or 6, wherein the carbon source is lupin hulls.
8. A process according to any one of the preceding claims wherein the mixed population of microorganisms comprises microalgae, yeasts, fungi, protists, microplankton and bacteria.
9. A process according to any one of claims 1 to 4, wherein the step d) of culturing occurs in saline conditions.
10. A process according to claim 9, wherein the step d) of culturing is conducted in sea water.
11. A process according to any one of the preceding claims, wherein the carbon to nitrogen ratio is in a range from about 2:1 to about 24:1 or is in a range from about 6:1 to about 18:1.
12. A process according to claim 11, wherein the carbon to nitrogen ratio is 6:1, 12:1, or 18:1.
13. A process according to any one of the preceding claims, further comprising the step of optimising the level of nutrients and the phosphorous to nitrogen ratio (P :N ratio) or the silicon.
14. A process according to any one of the preceding claims, wherein the carbon source is selected from a material which is utilisable by the bacteria.
15. A process according to any one of the preceding claims, wherein the step d) of culturing continues until most of the carbon source is broken down and converted into a flocculated biomass.
16. A process according to any one of the preceding claims, wherein the pH of the culture system is maintained at a pH of between 7.3 and 8.3.
17. A process according to any one of the preceding claims, wherein the source of microorganisms in step a) is a starter culture of one or more microorganisms which occur naturally in the water used in the culture system.
18. A process according to any one of the preceding claims, wherein the step of culturing is conducted over a period of from about 4 to 8 weeks.
19. A process according to any one of claims 1, 2 or 4, wherein the step of culturing is conducted in an open vessel which has a constant depth along the length of the vessel.
20. A microbial biomass produced by the process of any one of claims 1 to 19.
21. A feed product or feed ingredient comprising a microbial biomass which further comprises a mixed population of microorganisms including microalgae and bacteria, wherein the bacteria is present in an amount of from about 5 to 25wt% on a dry matter basis and microalgae is present in an amount from 10 to 80wt% on a dry matter basis.
22. A feed product or feed ingredient according to claim 21, wherein the bacteria is present in the microbial biomass in an amount from 5 to about 20wt% on a dry matter basis.
23. A feed product or feed ingredient according to claim 21 or 22, wherein the bacteria is present in the microbial biomass in an amount from about 5 to about 10wt% of the microbial biomass of claim 20.
24. Use of the microbial biomass of claim 20 or of the feed product of claim 21, 22 or 23 as an aquaculture feed or feed ingredient for an aquatic species.
25. Use according to claim 24, wherein the member of an aquatic species is selected from the group consisting offish, crustaceans or molluscs.
26. Use according to claim 25, wherein the fish is selected from the group consisting of Atlantic Salmon, barramundi and cobia trout; the crustaceans are selected from the group consisting of shrimps, lobsters and crabs; and the molluscs are selected from the group consisting of oysters, scallops and abalone.
27. A method of rearing an aquatic species comprising the steps of feeding an effective amount of the microbial biomass of claim 20 or of the feed product of claim 21, 22 or 23, to a member of an aquatic species.
28. A method according to claim 27, wherein the member of an aquatic species is selected from the group consisting of fish, crustaceans and molluscs.
29. A method according to claim 23, wherein the fish is selected from the group consisting of Atlantic Salmon, barramundi and cobia trout; the crustaceans are selected from the group consisting of shrimps, lobsters and crabs; and the molluscs are selected from the group consisting of oysters, scallops and abalone. |
MICROBIAL BIOMASS, FEED PRODUCT/ INGREDIENT AND PROCESSES FOR
PRODUCTION THEREOF
FIELD OF THE INVENTION
This invention relates to a microbial biomass, a feed product or ingredient for aquatic species, processes for producing a microbial biomass, processes for producing a feed product or ingredient, methods of rearing an aquatic species using the microbial biomass and the feed product or ingredient and uses of the microbial biomass and the feed product or ingredient of this invention. In particular, this invention relates to a microbial biomass, a feed product or ingredient, processes for producing a microbial biomass and a feed product or ingredient, methods of rearing an aquatic species using the microbial biomass and the feed product or ingredient and uses of the microbial biomass and the feed product or ingredient which utilise a mixed population of microorganisms comprising microalgae and bacteria.
BACKGROUND OF THE INVENTION
Microalgae are used in aquaculture as feeds for molluscs, crustaceans and some fish species, and for zooplankton used in aquaculture food chains. In the Australian context, microalgae therefore have a key role in the larval production of Pacific and Pearl oysters, prawns, barramundi and juvenile abalone, as well as other emerging species. Over the years, several hundred microalgal species have been tested as food, but probably less than twenty have been successful and have widespread use. Microalgae must possess a number of key attributes to be useful aquaculture species. They must be of an appropriate size for ingestion (e.g. from 1 to 15 microns for filter feeders) and readily digested. They must have rapid growth rates, be amenable to mass culture, and also be stable in culture under any fluctuations in temperature, light and nutrients that may occur in the culture systems. Finally, they must have a good nutrient composition, including an absence of toxins that might be transferred up the food chain.
Microalgal species can vary significantly in their nutritional value, and this may change under different culture conditions. Nevertheless, a carefully selected mixture of microalgae can offer an excellent nutritional package for larval animals, either directly or indirectly (through enrichment of zooplankton). Microalgae that have been found to have good nutritional properties — either as monospecies or within a mixed diet — include C. calcitrans, C. muetteri, P. lutheri. Isochrysis sp. (T.ISO), T. suecica, S. costatum and Thalassiosira pseudonana.
In general, microalgae provide a rich source of protein, and have a well-balanced amino acid composition. While the gross composition of microalgae can influence nutritional value, it is the balance of other key nutrients that possibly have most influence. Polyunsaturated fatty acids (PUFAsX especially docosahexaenoic acid (DHA) 5 eicosapentaenoic acid (EPA) and arachidonic acid (AA) — which are known to be essential for various larvae — vary significantly between algal classes and algal species. While most species have moderate to high concentrations of EPA, relatively few are rich in DHA. Isochrysis sp. (T.ISO), Pavlova lutheri, Micromonas pusilla and Rhodomonas salina are examples of DHA-rich microalgae.
Typical systems used indoors for microalgal mass culture include carboys (10—20 L), polythene bags (100-500 L) and tubs (1000-5000 L). These are usually operated in batch or continuous mode. For larger volumes, out-door tanks or ponds are used, operated semi- continuously. Depending on their scale, hatcheries may produce between several hundred to tens of thousands of litres of algae daily. Cell densities range from 10 —10 cells per millilitre with these standard systems, and production costs can range from US$50— 200, or 20-50% of hatcheries' operating costs. There are clear economies of scale with algal production, so that production costs become especially significant to smaller hatcheries. Consequently, there has been much effort directed at more cost-efficient production systems.
One of the perceived difficulties with large volume, outdoor systems is that they are susceptible to microbial contamination. Microalgal monoculture can be obtained but this requires an extreme culture environment, such as high salinity and/or high alkalinity. By contrast, indoor, closed systems allow for unialgal culture but are too expensive as a source of microalgae for the aquaculture industry and are more suited to high value applications such as pharmaceutical production.
Accordingly, there is a need for an improved, economical, large-scale process for producing microalgal biomass that can be harvested for use as an aquaculture feed.
The Applicant has found that culturing a mixed community of microorganisms under conditions where the growth of both microalgae and bacteria are encouraged results in a microbial floe having improved properties as a feedstuff for use in aquaculture. For example, shrimp and lobster fed on a diet supplemented with the microbial floe exhibited increased growth rates compared to shrimp and lobster fed on a conventional diet. By contrast to previous methods for producing microalgal biomass for aquaculture, bacterial growth is actually encouraged by the addition of a carbon source which is utilisable by the bacteria.
Accordingly, the resulting microbial floe contains a significant amount of bacterially-derived biomass. Previously, bacteria would have been considered as a contaminant.
BRIEF SUMMARY OF THE INVENTION
Accordingly, the present invention provides a process for producing a microbial biomass comprising: a) providing a mixed population of microorganisms comprising microalgae and bacteria; b) adding a carbon source to the mixed population of microorganisms; c) adding a nitrogen source to the mixed population of microorganisms; d) culturing the mixed population of microorganisms under conditions suitable for the growth of both the microalgae and bacteria to form a microbial biomass; and e) harvesting the microbial biomass.
It has been found by the Applicant that the yield of the microbial floe is improved when the carbon: nitrogen ratio in the culture medium is between particular ratios (6:1 to 18:1). The mixed population of microorganisms may also comprise microalgae, yeasts, fungi, protists, microplankton and bacteria.
In one embodiment, the carbon source is low value agricultural waste, which provides further cost savings in producing the microbial floe.
The culturing may take place in an open system such as a tank, pond or raceway, as opposed to a closed system such as a bioreactor. Previously, such open systems have been considered an undesirable means for obtaining microalgae for aquaculture and other uses due to the possibility of bacterial contamination. However, since the process described herein is based on encouraging bacterial growth in addition to microalgal growth, so-called bacterial "contamination" does not present a problem.
In the process of this invention, the culturing step d) may be conducted in an open vessel. In particular, the step d) of culturing may be conducted in open systems including but not limited to tanks, raceways or ponds.
When the microbial biomass is cultured in ponds, the ponds may be lined with one or more polymer sheets. The polymer sheets may be formed from a suitable material including but not limited to high density polyethylene (HDPE). However, the ponds may also be partially lined with polymer sheets around the edges and bank. The ponds may also remain unlined.
When the microbial biomass is cultured in raceways, the raceways may be lined with one or more polymer sheets. The polymer sheets may be formed from a suitable material including but not limited to high density polyethylene (HDPE). The raceways may also be located inside a structure which shields the raceways from wind and rain and which allows full penetration by sunlight.
When the microbial biomass is cultured in tanks, the tanks may be located inside a structure which shields the tanks from wind and rain and which allows full penetration by sunlight. The preferred depth of water in the culture system may be from 0.5 to 3m, 1 to 2.5m, 1 to 1.5m, 1 to 1,25m or about Im. However, the biomass may be cultured in shallower or deeper water.
In the process of this invention, the step d) of culturing may be conducted in saline conditions. The step d) of culturing may occur in saline conditions including but not limited to sea water, waste saline from a desalination plant, inland saline waters and the like. Where the step d) of culturing is conducted in sea water, the saline water may be seawater with a salinity ranging from 5 to 60ppt, 10 to 50 ppt, 15 to 40 ppt, 20 to 35 ppt or 30 to 35 ppt. However, this should not limit the step of culturing a biomass to within these salinities, as it may also be cultured in water with lower or higher salinities.
The process of this invention also comprise a step of mixing vigorously the mixed population of microorganisms comprising microalgae and bacteria in the presence of the carbon source and the nitrogen source to maintain flocculated particles of the microbial biomass in suspension. The flocculated particles of the microbial biomass (microbial floe) in suspension may also comprise a mixture of microalgae, bacteria, yeasts and fungi, organic detrital material, protists and other microorganisms.
The process of this invention may further comprise a step of drying the harvested biomass to form a feed product or feed ingredient.
The carbon source used in this invention may be a carbon source which is utilised by the bacteria in the process of this invention. In one aspect of this invention, all or most of the carbon source may be utilised by the bacteria. The carbon source may also be utilised by the bacteria and/or microalgae. The carbon source may be selected from the group consisting of waste, high volume, low value agricultural material and agricultural waste. The low value agricultural material may include products, by-products or waste streams from the processing of sugar cane (including molasses), rice, wheat, triticale, corn, sorghum, tapioca, oilseeds (including canola meal and lupin hulls), and elevator dust from grain handling facilities. Additional sources of both carbon and nitrogen could include production waste from feed
mills and distillery spent grain products. The carbon source in this invention may also be a material which has been milled or sieved. The carbon source may in a specific example be the hulls from lupins, canola, peanuts or other oil-seeds which have been milled or sieved to small particle size so that the particles would pass through a screen with a 2 mm mesh size.
The nitrogen source may be any economically and environmentally suitable product such as urea, ammonia, ammonium nitrate, ammonium phosphate fertilisers, organic nitrogen sources including discharge water from aquaculture ponds. Additional nitrogen may be added to the culture system. The concentration of total nitrogen in the culture system at the start of culture may be between 10 and 30 mg/L, or about 20 mg/L. hi a second aspect of this invention, there is provided a microbial biomass produced by the process of this invention and in particular the first aspect of this invention.
In a third aspect of this invention, there is provided a feed product including a mixed population of microorganisms including microalgae and bacteria wherein the dry weight ratio of bacteria to microalgae is between about 20:1 to about 0.4 to 1. The quantification of the microalgae may be based on the chlorophyll a content of the microbial biomass and that of the bacteria may be based on the muramic acid content. hi another aspect of this invention, there is provided a feed product or feed ingredient including a mixed population of microorganisms including microalgae and bacteria, wherein the bacteria is present in an amount of from about 5 to about 25wt% on a dry matter basis and microalgae is present in an amount of from 10% to about 80wt% on a dry matter basis. The feed product or feed ingredient may also comprise bacteria in an amount of from about 5 to about 20wt% on a dry matter basis. The feed product or feed ingredient may comprise at least 5 to 20 wt% of the microbial biomass of this invention.
Where the microbial biomass is used as a feed ingredient to form a feed product, it may be mixed with a binding agent (such as gluten, alginates or starch), an additional source of protein (such as fish meal, squid meal, krill meal, soybean meal, lupin meal, gluten meal), an ingredient rich in carbohydrate, specifically rich in starch, (such as wheat flour, rice bran, tapioca, rice flour, maize (or corn) flour), lipid sources (such as fish oil, squid oil, krill oil, vegetable oils, soybean oil, canola oil, soybean lecithin), a mixture of vitamins appropriate for the intended species, a mixture of minerals appropriate for the intended species, and other nutritional supplements.
In a fourth aspect of this invention, there is provided a use of the biomass of this invention or of the feed product of this invention in an amount effective to provide nutrition to a member of an aquatic species.
In a fifth aspect of this invention, there is provided a method of rearing an aquatic species comprising the steps of feeding an amount of the biomass of this invention or the feed product of this invention to a member of an aquatic species, said amount being effective to provide nutrition to said member of the aquatic species.
The member of an aquatic species may be selected from the group consisting of fish, crustaceans and molluscs. The fish may be selected from the group consisting of Atlantic salmon, barramundi and cobia trout The crustaceans may be selected from the group consisting of shrimp, lobsters and crabs. The molluscs may be selected from the group consisting of oysters, scallops, and abalone.
This invention results from the finding that products derived from the bioconversion of carbon sources via non-axenic multi-species cultivation of aquatic micro-organisms produce useful food, feed ingredients and bioactive compounds for aquaculture species and other livestock.
The process of this invention may also include a step of optimising the level of nutrients and the carbon to nitrogen ratio (C:N ratio) of readily utilizable carbon source and nitrogen for the production of the microbial biomass.
The nutrients which may be included in the step of optimising the level of nutrients are selected from phosphates, silicates and mixtures thereof. The phosphates may be selected from KH 2 PO 4 , superphosphate, double superphosphate, triple superphosphate, mono ammonium phosphate, diammonium phosphate, rock phosphate and Agras.
The silicate concentration of the culture system water may be adjusted to range from 20 mg/L to 163 mg/L silicate. The ratio of silica to nitrogen may be not less than 1.5:1.
KH 2 PO 4 may be added in an amount to provide a P:N ratio of from 2:1 to 20:1, 3:1 to 18:1, 4:1 to 16: 1, 4:1 to 14:1, or 5:1 to 10:1. Other P:N ratios maybe 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, 6.0:1. The phosphorous concentration in the water of the culture system may be present in proportion to the nitrogen content. The P:N ratio may be about 5.8:1 (P:N). The phosphate concentration of the culture water may be increased to this level by the addition of a phosphate containing fertiliser, for example mono ammonium phosphate. The phosphate concentration of the culture system water may be adjusted to be in the range from 90 mg/L to less than 710 mg/L.
The silicates may be selected from sodium silicate, sodium metasilicate (Na2Siθ 3 .5H 2 O), waterglass, and potassium silicate.
The silicate source may be added in an amount to provide a Si:N ratio of from 1:1 to 5:1, 1:1 to 4.5:1, 1:1 to 4.0:1, 1:1 to 3.5:1. 1:1 to 3.0:1, 1:1 to 2.5:1, 1:1 to 2.0:1, 1: 1 to 1.5:1.
The step of optimising the level of nutrients and the C:N ratio in the process of this invention may also encompass levels of the nitrogen source to levels as low as 5 mg L "1 and any utilizable carbon source, including waste from any agricultural crop. The C:N ratio may range from 2:1 to 24:1, 3:1 to 20:1, 4:1 to 18: 1, 5:1 to 18:1, or 6:1 to 18:1. Other C:N ratios may be 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1 and 24:1. Some specific examples of C:N ratio for this invention include 6:1, 12:1 and 18:1. We have also found that the yield of the microbial floe is improved when the carbon:nitrogen ratio in the culture medium is between particular ratios (6:1 to 18:1).
The process of this invention may also include a step of optimizing the surface area to volume ratio of the carbon source by milling and sieving to increase the efficiency of bioconversion to useful food or feed ingredients.
The products of the process of this invention and the feeds of this invention include yields of dry microbial biomass from 0.1 kg per 2.4 t tank to 1.1 kg per 2.4 t tank, with the greatest amount of dry microbial biomass and microbial protein produced from fine (<710μm) organic carbon material (See Example 2).
The wet microbial biomass may be dried by suitable means such as air drying or freeze drying. The water content of the dried microbial biomass may be reduced to less than 15% or less than 10%. Samples of the dried biomass harvested from each culture system may be weighed and analysed to determine dry matter, crude protein, total lipid and ash content using standard methods of analysis.
The dried microbial biomass may be included in a feed formulation at levels of from 5 to 15% or from 5 to 10% of the ingredient mixture. The dried microbial biomass may be used in the same way as any other dry feed ingredient.
The preferred usage of the biomass may firstly be as an animal feed additive; secondly as an aquatic animal feed additive; and thirdly as an additive in feeds for molluscs, crustaceans and fish.
The source of microbial organisms in the step of culturing may include a starter culture or one or more microorganisms which are present and that naturally occur in the water used in the culture system. The step of culturing may utilise whatever microbial community is present in the water that is used to fill the ponds, raceways or tanks. This community may become the microbial starter culture for the method of this invention. The water used to fill the culture ponds, raceways or tanks may be raw, unfiltered seawater drawn from any water source including but not limited to oceanic or estuarine waters, waste water from aquaculture ponds, or recycled water from a previous culture after the microbial floe has been harvested.
The starter culture of microbial organisms may also be supplemented with a natural population of microalgal and/or bacterial cultures. Additional starter culture comprising water drawn from a source rich in microalgae and bacteria may be added to the water in the step of culturing to supplement the existing microbial community. A particular group of microalgae or bacteria may also be added to the mixed microbial community in the culture system to speed up or improve the quality or quantity of microbial biomass production. The growth of undesirable species of microalgae (for example, a blue-green algae) in the culture system may be inhibited in the process of this invention by performing additional steps. These additional steps include changing the level of aeration, discharging surface water with the entrained undesirable microalgae, changing the pH, alkalinity, or the concentration of phosphate or silicate in the culture system.
When supplementing with a microalgal culture, the microalgae may be diatoms. When supplementing with bacterial culture, the culture may be nitrifying bacteria.
The water source used to fill the culture system may contain elevated levels of nutrients, particularly nitrogen in the form of ammonia, nitrate, nitrite and nitrogen-containing organic compounds such, as but not limited to protein and amino acids.
The step of culturing in the process of the invention may include mixing to maintain the particulate material in suspension, including flocculated material. The mixing of the water is to aerate it and ensure that anaerobic areas do not form on the bottom of the culture system. The mixing may be achieved by suitable means including for example aerators selected from the group consisting of paddle wheel aerators, air jet aerators, compressed air devices and airlift devices.
The process of this invention may also comprise maintaining the pH of the culture system in a range of from 7.3 to 8.3. The pH may be raised by suitable basic materials such as, but not limited to a member selected from the group consisting of hydrated lime, Aglime, dolomite and sodium carbonate.
The step of culturing the microbial biomass may continue until most of the carbon source has been broken down and converted into the flocculated biomass. This may be determined by microscopic examination of the biomass. The period of time hi which the step of culturing may take place may range from about 4 to about 8 weeks, depending on the economics of production, the carbon source being used and the particular conditions under which the culturing takes place.
The harvesting step of the process of this invention may comprise passing the contents of the culture system through a filtration device. The filtration device may be a screen filter,
such as a Baleen filter, or a continuous flow centrifugal device to concentrate the microbial biomass. The liquid which has passed through the filter or which has passed through the centrifuge may be returned to a culture pond, raceway or tank to provide the water, some nutrients and starter culture for the next batch of microbial biomass.
The harvested biomass may be pressed to remove more of the retained water and then rapidly dried. The drying may be carried out rapidly under a high air flow at moderate temperature. The temperature may range from 40 to 80° C. Alternatively, it may be dried under high airflow at a lower temperature, 40° C 5 or it may be dried within 12 hours, with or without decomposition. The dried product may contain less than 10% moisture.
The microbial biomass may also comprise a minimum bacterial content of 5% of the dried biomass. Detailed Description of the Preferred Embodiments
Examples
Example 1 (Culture of Microbial biomass)
The first experiment was carried out to obtain an indication of the optimum level of nitrogen and a readily utilizable carbon source for the production of a microbial biomass. The experiment comprised of twelve treatments each with duplicate tanks. The treatments comprised four concentrations of nitrogen (N) each in 12 tanks (Table 1). The source of the nitrogen was urea fertiliser. With each concentration of N, organic carbon, in the form of tapioca starch, was added to duplicate tanks to give C:N ratios of 6:1, 12:1 and 18:1 (Table 1). The tanks used for culturing the microbial biomass were circular fibreglass tanks (2450 L working volume) situated in a horticultural tunnel house that allowed rrήnunal attenuation of natural light and enabled the water temperatures to be maintained between 25 and 33°C.
The tanks were filled with filtered seawater (20 μm) five weeks prior to the start of the experiment. Additional micro organisms were added to each tank but adding 20 L of unfϊltered seawater collected from Cleveland Point, Moreton Bay, Queensland. The water in the tanks system was circulated through a mixing tank and pumped back into the tanks so that there was essentially a single mass of water. A microalgal bloom was encouraged through the addition a small amount of nutrient to each tank: 0.53 g urea, 0.1843 g KH 2 PO 4 and 2.8 g of Na 2 Siθ 3 .5H 2 O. Once the bloom was established, the circulation was stopped and the allocated treatments of urea, tapioca starch were added. At the same time, equal amounts of phosphate (in the form of KH 2 PO 4 ) and silicate (Na 2 SiO 3 .5H 2 O ) were added to all tanks. The KH 2 PO 4
was added to the tanks to provide a P:N ratio of 5.8:1, and the Na 2 SiOsJH 2 O was added to give a Si:N ratio of 1.5:1.
The nutrients and particulate material in the tanks was kept mixed and in suspension through vigorous aeration and the use of an air-lift device. Aiter 37 days, the microbial biomass was harvested from one tank at a time. This operation took three days. The aeration was first turned off and the microbial biomass and particulate material was allowed to settle. After one hour, the water was syphoned off through a filter bag from close to the surface until settled biomass was reached. The remaining water containing the bulk of the biomass was then passed through the filter bags. When the flow rate of water through a filter bag became unacceptably low, the bag was wrung out and the filtered biomass transferred into a plastic bag that was immediately placed under refrigeration. This process was continued until all of the water in the tank had been filtered.
The wet microbial biomass was weighed and spread on stainless steel trays and placed in a high airflow drying oven at 40°C for 2 days. The water content of the dried microbial biomass had been reduced to <10%. The dried biomass harvested from each tank was weighed and analysed to determine dry matter, crude protein, total lipid and ash content using standard methods of analysis (Table 2). The results indicated that the yield of biomass increased with increasing N input, but that the retention efficiency was optimal with 20 mg L " 1 of input N. A C:N ratio of 12:1 appeared to provide optimal efficiency for the production of the biomass, though a ratio of 18: 1 tended to produce more protein.
Table 1. Treatments and amounts of urea, tapioca starch, KH2PO 4 and Na 2 SiO 3 -SH 2 O added to 2450 L tanks used to culture microbial biomass
Nitrogen Treatment Weight of Weight of Amount of Amount of
Input C:N ratio urea (g) tapioca (g) KH 2 PO 4 Na 2 SiO 3 .5H 2 O
(mg L- 1 ) (g) (R)
5 6:1 26.3 170 127.3 56.8
5 12:1 26.3 330 127.3 56.8
5 18:1 26.3 500 127.3 56.8
10 6:1 52.5 330 254.6 113.6
10 12:1 52.5 660 254.6 113.6
10 18:1 52.5 990 254.6 113.6
20 6:1 105.0 660 509.2 227.1
20 12:1 105.0 1320 509.2 227.1
20 18:1 105.0 1980 509.2 227.1
40 6:1 210.0 1320 1018.5 454.2
40 12:1 210.0 2650 1018.5 454.2
40 18:1 210.0 6970 1018.5 454.2
Table 2. Yield from each tank and composition of dried microbial biomass cultivated in Experiment 1. Crude protein, total lipid and ash contents reported on a Dry Matter basis.
Nitrogen Treatment Dried Dry Crude Total Ash
Input C:N ratio weight of Matter Protein lipid (g kg- 1 )
(mg L- 1 ) biomass (gkg 1 ) (g kg 1 ) (g kg 1 )
(g)
5 6:1 230 92.0 14.1 4.05 54.4
5 6:1 306 92.8 15.6 3.53 53.6
5 12:1 400 93.4 16.9 2.99 54.3
5 12:1 230 92.6 15.1 3.58 57.1
5 18:1 520 93.7 12.2 1.96 52.0
5 18:1 388 94.0 11.8 2.39 41.6
10 6:1 515 95.1 19.5 3.54 46.0
10 6:1 270 86.6 15.3 3.02 62.5
10 12:1 355 91.4 14.9 3.29 63.7
10 12:1 170 92.3 19.8 4.19 56.2
10 18:1 710 93.8 14.7 2.91 53.4
10 18:1 370 92.3 19.6 3.14 55.6
20 6:1 1011 91.3 15.5 2.81 65.0
20 6:1 657 91.0 16 A 3.45 62.3
20 12:1 1182 92.4 17.6 3.33 62.2
20 12:1 1096 93.4 17.6 4.78 61.6
20 18:1 839 94.8 20.8 4.19 55.9
20 18:1 1230 93.1 16.8 4.00 62.7
40 6:1 1760 91.0 13.3 2.73 69.4
40 6:1 1395 89.2 11.5 1.83 71.7
40 12:1 1610 91.4 13.6 2.56 68.3
40 12:1 1300 93.3 17.2 2.72 65.1
40 18:1 1710 91.5 17.1 3.27 64.0
40 18:1 1610 92.0 15.8 3.17 64.8
Example 2. (Culture of microbial biomass using lupin hulls)
This example was carried out to assess the use of lupin hulls as a low cost carbon source for the culture of microbial biomass and to compare the yield with that obtained using tapioca starch. The lupin hulls were a by-product of the processing of lupin seeds to obtain lupin kernels. A sample of the lupin hulls was passed through a hammer mill to reduce their size and hence provide a larger surface area to volume ratio. The expectation was that this would increase the rate of bioconversion of the lupin hull material by the microbial community in the culture tanks. The milled material appeared to comprise two broad size fractions and was sieved with a 710 μm screen to separate them. These two fractions provided two of the carbon sources. Analysis of the fractions revealed slight differences in the proximate
composition (Table 3). Refined lupin fibre, a product prepared commercially and used by the food industry as a fibre additive, was included as an additional treatment.
The experiment comprised six carbon source based treatments with duplicate tanks assigned to each treatment in a fully randomised design. The treatments were: (A) Control, with no added carbon source, (B) tapioca flour, (C) refined lupin fibre, (D) fine lupin hull material, (E) course lupin hull material, and (F) unmilled lupin hulls. The tanks used for culturing the microbial biomass were circular fibreglass tanks (2450 L water volume) that were located down one side of a horticultural runnel house. The tunnel house allowed minimal attenuation of natural light and enabled the water temperatures to be maintained between 25 and 33°C. Each of the tanks was filled with a mixture of unfiltered seawater (20 L) and filtered seawater four weeks prior to the start of the experiment. Nutrients were added to initiate a microbial starter culture (0.53 g urea and 0.1843 g KH 2 PO 4 to each tank).
The water was circulated through a mixing tank and pumped back into the tanks so as to ensure that the microbial community and the concentration of nutrients was the same in all tanks prior to the start of the experiment. Once a bloom was established, the circulation was stopped and the allocated treatments of urea and carbon source were added to the individual tanks.
Urea was added to provide 20 mg L "1 of N (105 g of urea per tank), and the carbon sources were added to provide a C:N ratio of 12:1 (1.323 kg per tank). In addition KH 2 PO 4 was added to all tanks to give a P:N ratio of 5.8:1 (509 g per tank). The nutrients and particulate material in the tanks was kept mixed and in suspension through vigorous aeration and the use of an air-lift device in each tank.
After 39 days, the microbial biomass was harvested one tank at a time. This operation took two days. The aeration was first turned off and the microbial biomass and particulate material was allowed to settle. After one hour, the water was syphoned off through a filter bag collecting from close to the surface until settled biomass was reached. The remaining water containing the bulk of the biomass was then passed through the filter bags. When the flow rate of water through a filter bag became unacceptably low, the bag was wrung out and the filtered biomass transferred into a plastic bag that was immediately placed under refrigeration. This process was continued until all of the water in the tank had been filtered. The wet microbial biomass was weighed and spread on stainless steel trays and placed in a high airflow drying oven at 40 0 C for 2 days.
The water content of the dried microbial biomass had been reduced to <10%. The dried biomass harvested from each tank was weighed and analysed to determine dry matter,
crude protein, total lipid and ash content using standard methods of analysis (AOAC 1991) (Table 4).
The results indicated that the greatest concentration of crude protein was in the microbial biomass cultured using tapioca flour as the carbon source. However, this treatment produced the least amount of biomass.
The yield of wet biomass was variable with greatest amount of dry microbial biomass was produced in the tanks containing the fine lupin (<710μm) material. On average, the yield from this experiment was about 1 kg/ 2.4 tonne tank. The experiment demonstrated that lupin hulls were an effective carbon source for the culture of microbial biomass, and that finely grinding the hulls resulted in improved yield of microbial protein.
Table 3. Composition of carbon sources used for the culture of microbial biomass. Results reported on % DM basis, unless otherwise indicated. ADF= acid detergent fibre
Dry Crude Crude fat Ash. ADF matter* protein
Tapioca flour 86.5 0.0 0.0 0.2 0.5
Refined lupin fibre 91.3 6.1 1.9 2.7 64.8
Fine lupin hull 92.6 17.3 4.6 2.9 46.4
Coarse lupin hull 93.2 8.1 - 2.6 59.9
Unmilled lupin hulls 91.4 11.4 - 2.9 57.1
* %, as received
Table 4. Yield (air dried biomass) and composition of microbial biomass cultured using tapioca and lupin hulls as carbon sources. Results from duplicate tanks are reported. Composition data are expressed as %, on dry matter basis. ADF= acid detergent fibre
Yield Crude Total Ash ADF
(g/tank) protein lipid (%) (%)
(%) (%)
Control 322 34.8 6.2 28.4 19.7
Control 276 36.3 7.6 33.5 15.9
Tapioca flour 998 21.3 2.9 54.2 17.3
Tapioca flour 1014 21.4 3.0 53.6 19.2
Refined lupin fibre 1033 18.8 3.6 49.9 22.6
Refined lupin fibre 519 23.1 4.1 47.6 21.4
Fine lupin hulls 1124 22.1 3.2 52.9 17.5
Fine lupin hulls 119 26.3 3.5 48.6 17.2
Coarse lupin hulls 884 23.3 3.9 43.8 24.7
Coarse lupin hulls 1059 25.8 4.2 41.3 24.4
Unmilled hulls 994 32.5 4.5 37.5 21.8
Unmilled hulls 840 27.2 3.9 43.6 20.2
Example 3.
The objective of this study was to investigate the variability in the nutritional value of multiple cultures of microbial biomass when included in feeds for the black tiger shrimp, Penaeus monodon. The microbial biomass had been produced in 2500 L tanks at the CSIRO facility at Cleveland as outlined in Example 1 and 2. This study was carried out in a clear water aquarium system.
The experiment comprised of a 35-day feeding trial with a basal diet and a series of 15 diets each containing lOOg kg "1 of dried microbial biomass from a separate culture batch. The formulation of the basal diet and three of the test diets are given in Table 1 to illustrate the way the diets were formulated.
During formulation, the crude protein content and fat content (75 g kg "1 DM) were maintained at the same level across all diets, (420 g kg "1 and 75 g kg "1 DM, respectively). The dried microbial biomass was included in the test diets at the same level (100 g kg "1 ) with an adjustment to the amount of casern, mixed vegetable oil and wheat starch to balance the formulation. The weighed ingredients were thoroughly mixed in a planetary mixer before a volume of water equivalent to approximately 40% of the dry weight of ingredients was added, and mixed further to form a crumbly dough. The dough was extruded through the meat grinder attachment of a Hobart A-200 mixer (Hobart Corporation, Troy, OH, USA). The extruded, spaghetti-like strands (-3 mm diameter) were steamed for 5 min in an atmospheric steamer (Curtin & Son, Sydney, Australia), air-dried overnight in a forced-draught cabinet at 40 0 C and broken into pellets 5 to 8 mm long. The pellets were stored at -20 0 C until used.
Juvenile P. monodon were obtained from commercial shrimp farms in northern Queensland, Australia. They were held at the CSIRO Marine Research Laboratory, Cleveland in 2500 L tanks for about one week before being transferred to the smaller tanks used for the experiment. While held in the 2500 L tanks, the shrimp were fed twice daily with a commercial P. monodon feed (CP # 4004, CP Feeds, Samut Sakorn, Thailand). The tanks were supplied with flow-through, filtered seawater (salinity 32 to 36 %o) that maintained the temperature at 28 ± 0.5 0 C. For the growth response experiment, an array of circular, white polyethylene indoor tanks (120 L capacity, 600 mm diam.) was used. Each tank was supplied with filtered (10 μm), heated seawater flowing at a rate of 600 mL min "1 to maintain tank temperatures at 29.0 ± 0.5 °C, and provided with supplementary aeration from a single air- stone. Water temperatures were monitored daily and a 12 h light: 12 h dark photoperiod was maintained throughout the experiments.
Prior to the start of the experiment, the shrimp were individually weighed and sorted into size classes so that shrimp within a class had a weight range of no greater than 0.5 g. Shrimp of between 2.5 g and 3.6 g were used in this experiment. The shrimp were distributed among the array of tanks with six shrimp in each tank, such that the biomass in all the tanks was similar.
The shrimp were allowed to adapt to the tank conditions and the basal diet for 7 days before they were individually weighed again at the start of the experiment. At this weighing, only five shrimp were returned to each tank to further reduce the variability in the weight range of individual shrimp and the biomass among tanks (mean ± sd = 3.2 ± 0.30 g).
They were weighed again after 25 days and at the end of the experiment at 35 days. During the experiment, the shrimp were fed weighed allocations of their assigned feeds twice daily, nominally at 0830 and 1700 h. The tanks were cleaned daily in the afternoon and the amount of uneaten feed in the tank was noted using a scale of 0 to 4.
The following day's allocation of feed was adjusted according to this value, so as to minimise the amount of uneaten feed but also to ensure that growth was not limited by consistent underfeeding. Any dead or missing shrimp were replaced within 24 hours with tagged shrimp of similar size. Tagged replacement shrimp were used to maintain a constant stocking density in the tanks but were not included in the data used to analyse growth response or survival. Though individual weights were recorded, only the mean weight of untagged shrimp within each tank was used in the data analysis.
The results of the experiment are presented in Table 2. Survival was high across most treatments and the average for the experiment was 85%. The inclusion of microbial biomass in the feed resulted in a significant increase in growth for 11 of the 15 diets, hi no case was the growth obtained with a microbial biomass containing numerically less than that obtained with the basal diet. The average increase in growth obtained by including the microbial biomass in the feed was 35% (sd = 10.5%) over the growth obtained with the basal diet.
See Table 1 below. Ingredient composition of the basal diet and three of the test diets that illustrate the way the diets were formulated.
Table 1
Ingredient Basal Test 5-18 Test 10-6 Test 10-12
Fish meal 68% 340 340 340 340
Krill meal 20 20 20 20
Squid meal 50 50 50 50
Gluten (wheat) 50 50 50 50
Casein 31 21 14 14
Microbial biomass 0 100 100 100
Lecithin 10 10 10 10
Mixed veg. oil 11 9 8 8
Wheat starch 224 135 143 144
Flour 200 200 200 200
Aquabind 30 30 30 30
Cholesterol 1.0 1.0 1.0 1.0
Banox E 0.2 0.2 0.2 0.2
Carophyll Pink 8% 0.5 0.5 0.5 0.5
Vit C (Stay C) 1.0 1.0 1.0 1.0
Vit premix 2.0 2.0 2.0 2.0
TOTAL 1000 1000 1000 1000
Table 2. Biological response parameters of shrimp fed the test diets for 4 weeks. Benefit is the difference between the growth achieved with the test diet and the basal diet, expressed as a percentage of the basal diet. Initial weight of shrimp (mean± sd = 3.2 ± 0.3 g)
Final Growth Benefit Survival weight (g) (g/wk) (%) (%)
Basal 7.53 0.87 - 80
5-8 8.83 1.13 30 73
10-6 9.54 1.27 46 93
10-12 9.31 1.23 28 90
10-18 9.22 1.21 28 87
20-6 9.73 1.31 36 77
20-12 8.58 1.08 16 97
20-18 9.77 1.32 42 93
40-6 9.10 1.19 24 87
40-12 8.96 1.16 24 87
40-18 8.85 1.14 23 77
2-2 8.82 1.13 23 77
2-3 8.68 1.10 20 87
2-4 9.04 1.17 27 97
2-5 8.06 1.00 11 73
2-6 9.34 1.22 35 93 s.e.m. 0.416 0.083 2.6 5.3
Example 4.
The objective of this study was to measure the effect of the inclusion of dried microbial biomass in the feeds of black tiger shrimp, Penaeus monodon when prawns grown in a green water culture environment.
The experiment involved four diet-based treatments with 6 replicate 2500 L tanks assigned to each treatment. The diets comprised a basal diet and two diets containing dried microbial biomass at an inclusion level of 50 and 100 g kg "1 . The diets comprised of a base (900 g kg *1 ) that was the unprocessed feed ingredient mix of a commercial shrimp feed (Starter feed, Ridley AquaFeeds, Narangbar, QId, Australia) the balance (100 g kg "1 ) comprising either a mixture of casein, lupin bran and ground clam shell, and/or dried microbial biomass (See Table 1).
The weighed ingredients were thoroughly mixed in a planetary mixer before a volume of water equivalent to approximately 40% of the dry weight of ingredients was added, and mixed further to form a crumbly dough. The dough was extruded through the meat grinder attachment of a Hobart A-200 mixer (Hobart Corporation, Troy, OH, USA). The extruded, spaghetti-like strands (—3 mm diameter) were steamed for 5 min in an atmospheric steamer (Curtin & Son, Sydney, Australia), air-dried overnight in a forced-draught cabinet at 40 0 C and broken into pellets 5 to 8 mm long. The pellets were stored at -20 0 C until used.
The 24 x 2500 L fibreglass tanks were set up in a horticulture tunnel house. The tanks were filled with seawater, contained no sand substrate on the bottom and were provided with individual reticulated aeration. The water supply of all tanks was circulated in a semi-closed system as one body of water. Tank water temperature was maintained within a narrow range by heating the water with heat exchangers to ensure a minimum temperature 27.5°C. When required on hot, sunny days, a ceiling of shade-cloth was extended across the whole tunnel house to prevent the water temperature increasing above 33 0 C.
Two weeks prior to the start of the experiment, 20 L of unfiltered seawater, collected from Cleveland Point, Moreton Bay, Queensland, was put into each of the tanks and the tanks filled with filtered seawater from the laboratory supply. The tanks were then lightly fertilized with urea to establish a microalgal bloom. Throughout the experiment, the microalgal bloom and nutrient levels in the water were managed by discharging and replacing water as needed, emulating shrimp pond management practice.
Over 2000 shrimp of between 3.5 and 6.0 g were collected from a commercial shrimp farm in central Queensland (Seafarm, Cardwell, QId). The shrimp were weighed on site at the
prawn farm and placed in consignment boxes which were allocated to specific tanks. 75 shrimp were allocated to each tank, so that size composition and mean weight was similar and not significantly different among tanks (mean ± sd = 3.2 ± 0.3 g). On arrival at the CSER.0 facility at Cleveland, the shrimp were placed directly into the designated tanks and the experiment started.
The shrimp were fed 3 times daily (nominally at 0600, 1100 and 1700) with then- assigned feed. In each tank, all of the feed was placed on two feeding trays (300 mm diameter). The feeding trays were removed just prior to the next feeding and the amount of feed remaining was assessed. The shrimp were fed weighed rations to satiation as judged by the feed remaining on feeding trays. After 4 weeks, growth, FCR and survival was measured after draining each tank and recovering all of the shrimp. Water quality in all tanks was monitored twice daily at 0500, 1400 by measuring DO, temperature, pH, turbidity, salinity in all tanks using data logger (YSI). Weekly water samples were also taken for nutrient analysis (ammonia, dissolved nitrogen, nitrate). Ih addition fluorescence (chlorophyll) and light attenuation was measured.
The results are shown in Table 2. The shrimp growth rate of the shrimp was high and increased significantly with increasing inclusion of dried microbial biomass. Feed intake also increased with increasing level of dried microbial biomass and as a result, FCR' s remained relatively constant across treatments. Survival was high across all treatments with a mean survival of 93%. The results clearly demonstrate an increase in growth rate attributable to the inclusion of dried microbial biomass in the feed. Table 1. Ingredient and nutrient composition of test diets (g kg "1 .)
Ingredient Basal Test 5 Test 10
Shrimp feed mash (Ridley) 900 900 900
Dried microbial biomass 0 50 100
Lupin bran 20 10 0
Casein 30 15 0
Ground clam shell 50 25 0
TOTAL 1000 1000 100.0
Nutrient (DM basis)
Crude protein 479 472 469
Crude fat 71 73 73
Ash 129 132 141
Table 2. Initial weight and biological response parameters of shrimp fed the test diets for 4 weeks
Basal Test 5 Test 10 s.e.m.
Initial weight (g) 3.9 3.9 3.9 0.03
Final weight (g) 12.8 14.8 16.2 0.25
Growth (g/week) 1.56 1.92 2.16 0.04
Survival (%) 93.3 92.0 94.0 2.1
Feed allocation (g/tank) 1559 1938 2457 . 35
FCR 2.53 2.56 2.84 0.08
Advantages
Some advantage of this invention may be as follows:
1) Growth rates of shrimp reared in either clear water or green water and fed diets containing microbial biomass increased by about 35% (sd = 10.5%) over the growth obtained with the basal diet containing wild harvest fishmeal (See Examples 3 & 4 above);
2) The size of shimp at harvest would be larger and result in a higher price per kilo (up to 25% higher) than the equivalent biomass of smaller sized shrimp.
Modifications and variations such as would be apparent to a skilled addressee are deemed to be within the scope of this invention. It is to be understood that this invention should not be limited to the specific example(s) and embodiment(s) described above.