WO2017105231A1 - Compositions déshydratées de protozoaires et procédés d'obtention de celles-ci - Google Patents

Compositions déshydratées de protozoaires et procédés d'obtention de celles-ci Download PDF

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Publication number
WO2017105231A1
WO2017105231A1 PCT/NL2016/050874 NL2016050874W WO2017105231A1 WO 2017105231 A1 WO2017105231 A1 WO 2017105231A1 NL 2016050874 W NL2016050874 W NL 2016050874W WO 2017105231 A1 WO2017105231 A1 WO 2017105231A1
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Prior art keywords
protozoa
composition
drying
spray
dry
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PCT/NL2016/050874
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English (en)
Inventor
Wilhelmus Theodorus Antonius Maria De Laat
Valeska VAN BREUGEL
Martinus Adrianus Maria VAN IERSEL
Ap DE HAAN
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Ibema Biezenmortel B.V.
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Publication of WO2017105231A1 publication Critical patent/WO2017105231A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor

Definitions

  • the invention relates to the field of agriculture, more specific to the field of improving plant growth.
  • normally fertilising compositions are used which may profit from enrichment in or addition of certain micro-organisms, such as bacteria, archaea, fungi and protozoa.
  • certain micro-organisms such as bacteria, archaea, fungi and protozoa.
  • compositions of these micro-organisms need to be developed.
  • Increased plant growth due to protozoa-bacteria interactions in the rhizosphere is well documented (Clarholm, 1984, In: Klug, M.J., Reddy, C.A. (Eds.), Current Perspectives on Microbial Ecology, American Society of Microbiology, Washington, pp.
  • Protozoa and also nematodes
  • Bactozoa that feed on bacteria and fungi will excrete ammonia, amines and amino acids as they have a much higher C/N ratio than protein rich bacteria.
  • a legal regulation for biofertiliser composition and use is being prepared and herein it is suggested that protozoa should be part of biofertiliser preparations (A contribution to set legal framework for biofertilisers, Mini review by Malusa et al., 2014, Appl. Microbiol And Biotechnol. 98: 6599-6607).
  • protozoa Another useful application of protozoa is to minimize problems with human pathogenic bacteria such as E. coli, Salmonella and Listeria in substrates that are used for growing plants, such as often present in peat, coco, etc. due to grazing by protozoa.
  • Grazing protozoa may also be applied in the reduction or control of pathogens in food products such as in slaughter houses a role as indicated by Vaerewijk et al. (Microscopic and Molecular Studies of the Diversity of Free living Protozoa in Meat Cutting Plants,
  • the present inventors have been able to provide a process for industrial production of protozoa preparations, especially where these preparations can be provided in a storable, dried form.
  • the drying process is done through drying in (hot) air, such as spray-drying, fluid bed drying, combinations between fluid bed and spray-drying, belt drying, etc.
  • the invention is related to a method for obtaining a dry protozoa composition by drying a liquid protozoa composition in air.
  • the drying is performed through spray drying or flash drying.
  • said spray drying or flash drying is performed in the presence of a protecting agent, preferably wherein said protecting agent is selected from the group of hydroxyectoine, sugars like sucrose, glucose, trehalose, lactose, lactulose, maltose and maltodextrines, galacto- oligosaccharides and polyols like sorbitol, mannitol, and glycerol, and skimmed milk.
  • a carrier is added, preferably starch.
  • the spray or flash dried composition is granulated or agglomorated.
  • the method for air drying a protozoa composition comprises fluid bed drying or belt drying.
  • said liquid protozoa composition also comprises other micro-organisms, preferably fungi, yeast, bacteria and/or archaea.
  • the protozoa composition is filtered in order to be essentially free of bacteria, after which the composition is dried in a fluid bed dryer or belt dryer, more preferably the filter is a filter in the range of 1 to 20 mu, preferably 2 mu.
  • liquid protozoa composition is compost tea, preferably FytaforceTM.
  • a dry protozoa composition produced by any of the methods according to the invention.
  • Said dry protozoa composition is preferably packaged in a low oxygen environment, preferably in vacuum or under N2 or CO2.
  • Further part of the invention is a method for storing the dry packaged protozoa composition obtained as indicated above, wherein said composition is stored at temperatures below room temperature, preferably below 5 °C.
  • Also part of the invention is the use of the dry protozoa composition as obtained according to a method of the invention for improvement of plant growth, for addition to a soil fertiliser, preferably a biofertiliser, or for use in sanitation.
  • protozoa are herein defined as a group of micro-organisms, comprising flagellates, ciliates and/or amoebae, preferably comprising organisms belonging to the groups of Amoebozoa, Rhizaria, Alveolata, Stramenopiles, Excavata and/or Ophisthokonta, more preferably comprising organisms belonging to the groups of Cercozoa, Foraminifera, Conosa, Lobosa, Labyrinthulea, Ochrophyta, Discoba and/or Rotifera
  • protozoa have been found to be very advantageous in agricultural applications, many fertilising compositions have little or low amount of protozoa.
  • Compost and compost tea are the main commercial sources of protozoa.
  • Compost consists mainly of woody biomass, stones, sand etc. and the number of protozoa is low in comparison to the number of bacteria and other micro-organisms.
  • a method to increase the amount of these micro-organisms and also the relative amount of protozoa is the production of so-called compost tea.
  • Compost tea is a water extraction of compost.
  • the micro-organisms present in the compost are multiplied by adding selective nutritive substrata. As the amount of microbes in the extraction increases, the level of dissolved oxygen decreases and thus air is constantly bubbled through the system to keep the extraction aerobic. After 48 hours this brewing process is complete and the compost tea then consists of nutritive materials (organic compounds, micro and macro elements) and a wide variety of beneficial bacteria, fungi, protozoa and nematodes.
  • Compost teas are sieved after brewing using 100 ⁇ , 250 ⁇ or 400 ⁇ filters to remove large parts and allows for the application of compost tea through spraying systems and through drip irrigation systems. This makes application of protozoa through compost tea easier than compost and more favorable than compost.
  • One of the goals of the present invention therefore is to provide a dried protozoan composition which is readily applicable in agriculture and the control of levels of bacteria, archaea, fungi, yeasts, etc. through
  • a mixed composition of protozoa is preferred, i.e. a protozoa composition comprising more than two, preferably more than three, more preferably more than four, more preferably more than five, most preferably a multitude (i.e. more than ten) different species.
  • Freeze-drying is a relatively expensive drying technique which is mainly used for storage of (pure) protozoan cultures in the laboratory or with culture collection institutes like CCAP (culture collection of protozoa and algae) or ATCC (American Type Culture
  • the present invention shows that it is possible to dry (large volumes of) protozoa compositions through fluid bed drying or via spray drying in a commercially attractive way.
  • the protozoa composition may consist of a pure culture of protozoa, but - as has been indicated above - it is preferred to have a composition that comprises more than one species of protozoa.
  • composition may also comprise other micro-organisms, such as fungi, yeasts, archaea and bacteria.
  • a method to obtain a composition containing a high variety and number of protozoa is to cultivate these in a liquid compost extract (compost tea), sieve the large particles, such as remaining plant materials, remove the sand and then to preserve the protozoa by drying methods as provided herein, so the protozoa are marketable.
  • Another method is to cultivate the protozoa by growing a certain bacterium in an axenic culture, feed it to an axenic culture of protozoa, and subsequently harvest the protozoa by centrifugation, microfiltration or passing through a sieve and subsequent drying the protozoa as a monoculture.
  • the protozoa may be produced as actively growing cells or as vegetative cells, but the compositions may also contain the protozoa in a cyst and/or spore-form.
  • the inventors of the present invention have unexpectedly discovered that protozoa in protozoa compositions (see the experimental section) show excellent survival and viability rates after drying with air, preferably with warm air of a temperature 30 °C or more . It is submitted that the temperature should not be too high in order to maintain the viability of the protozoa. Therefore, a temperature between 30 and 180°C, more specifically between 30 and 135°C is preferable. Further, preferred drying methods are spray-drying and fluid bed drying. The absolute amounts of protozoa show a rapid increase after rehydrating spray-dried compositions demonstrating excellent survival and viability. When compost tea is used as the material from which the protozoa composition is derived, it is possible to spray dry this compost tea directly. Additionally protozoa can be concentrated by centrifugation, microfiltration or passing through a sieve prior to spray drying.
  • spray drying is the production of highly dispersed droplets from the liquid and drying these to a powder from a liquid feed by evaporating the solvent. This is achieved by mixing a heated gas with an atomized (sprayed) fluid (in this case the compost tea) of high surface-to-mass ratio droplets, ideally of equal size, within a vessel (drying chamber), causing the solvent to evaporate uniformly and quickly through direct contact.
  • atomized (sprayed) fluid in this case the compost tea
  • a vessel drying chamber
  • spray drying offers a very flexible control over powder particle properties such as density, size, flow characteristics and moisture content.
  • Spray drying consists of the following phases:
  • This feed composition preferably is a homogenous, pumpable and free from impurities solution, suspension or paste. In the case of compost tea, it means that larger impurities, such as remaining organic debris should be filtered of before entering the solution as a feed for spray drying.
  • Drying A constant rate phase ensures moisture evaporates rapidly from the surface of the particle. This is followed by a falling rate period where the drying is controlled by diffusion of water to the surface of the particle. Separation of powder from moist gas: To be carried out in an economical (e.g. recycling the drying medium) and pollutant- free manner. Fine particles are generally removed with cyclones, bag filters, precipitators or scrubbers.
  • Pressure nozzle atomization Spray created by forcing the fluid through an orifice. This is an energy efficient method which also offers the narrowest particle size distribution.
  • Two-fluid nozzle atomization Spray created by mixing the feed with a compressed gas. Least energy efficient method. Useful for making extremely fine particles.
  • Centrifugal atomization Spray created by passing the feed through or across a rotating disk. Most resistant to wear and can generally be run for longer periods of time.
  • the drying in the spray dryer takes place at elevated
  • temperatures where the temperature of the heated gas at the start of the process is held at a temperature of 100 - 180°C, preferable at a temperature of 110 - 150°C, more preferably at about 135°C.
  • temperature at the outlet of the spray drier which can only be controlled by adjusting the temperature of the gas and the temperature of the feed. Adjusting these to reach an outlet temperature of 50 - 110°C, more preferably 55 - 75°C is preferred.
  • the feed that contains the protozoa fluid composition may be supplied with additives.
  • additives may comprise a desiccation protectant such as hydroxyectoine, sugars hke sucrose, glucose, trehalose, lactose, lactulose, maltose and maltodextrines, galacto-oligosaccharides and polyols hke sorbitol, mannitol, and glycerol.
  • a desiccation protectant such as hydroxyectoine, sugars hke sucrose, glucose, trehalose, lactose, lactulose, maltose and maltodextrines, galacto-oligosaccharides and polyols hke sorbitol, mannitol, and glycerol.
  • the concentration of desiccation protectant preferably varies from 1 - 10% in the second feed liquid, preferably about 2%.
  • the ratio between compost tea feed and second feed liquid may vary from 10 : 1 - 1 : 100 (on a dry weight basis).
  • Such a carrier compound may be added at a concentration of 1 - 20%, preferably 5 - 10%, more preferably about 8%.
  • the compost tea may preferably be the product FytaforceTM that is commercially available from SoilTech, a company in Biezenmortel, The Netherlands.
  • This product contains typically 2.10 6 protozoaml of a diverse range of protozoa which are normally present in compost and healthy soils.
  • Table 1 gives an overview of this wide diversity protozoa encountered in FytaforceTM as determined by sequence analyses of the V9 region of the 18S rRNA gene.
  • FytaforceTM additionally contains at least, 4.10 7 /ml yeasts and fungi and 1.10 9 /ml bacteria at a pH of 6.0 - 8.0. It further contains 0.5 - 0.7 gr/1 nitrogen and 0.7 - 1.3 g/1 phosphorus (in the form of P2O5).
  • This product is advantageously suited as a biofertiliser, i.e. a composition that may be used in organic agriculture.
  • Compounds like sucrose, starch and maltodextrins would be suitable for this purpose.
  • water binding compounds such as PVP (polyvinylpyrrolidone (Kadam et al, Granulation of Bioproducts, CRC Press, 1990) can be used to improve storage stability.
  • Flash drying is the process of drying particulate matter by exposing it briefly (typically a few seconds) to a high temperature gas stream, resulting in a rapid rate of evaporation without excessive heating of the product.
  • the process is used in various industries, such as the food and wood processing industries, and considerable literature exists on the design and modelling of the process (El-Behery, S. et al., 2009, World Acad. Sci. Eng. Technol.
  • the powder may be granulated or agglomerated to prevent dusting during further processing.
  • the powder After the drying the powder may be packaged and stored for extended periods of time without losing its composition and its intrinsic properties that make it excellently suitable for agricultural fertilising purposes.
  • Packaging is preferably done under low oxygen or oxygen free conditions such as vacuum, nitrogen (N2) or carbon dioxide (CO2).
  • a reduced pressure such as a partial vacuum with more than 100 mbar underpressure, preferably more than 200 mbar underpressure more preferably more than 300 mbar underpressure, more preferably more than 800 mbar underpressure and most preferably more than 900 mbar underpressure, is advantageous for packaging the protozoa composition of the invention.
  • packaging should take place under conditions where the amount of oxygen is (far) below the oxygen content in air and also where the pressure is below air pressure.
  • the oxygen is (far) below the oxygen content in air and also where the pressure is below air pressure.
  • concentration is less than 18%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5% of the total atmosphere. Most preferable is packaging in absence of oxygen, such as under an N2 or CO2 or mixed N2-CO2 atmosphere. .
  • the packaged protozoa composition should preferably be as dry as possible.
  • the content of dry matter should be > 90%, preferably >92%, even more preferable >95% of the total composition to be packaged.
  • the packaged protozoa composition should be preferably stored at temperatures below room temperature. More preferable below 5 °C.
  • any liquid protozoa composition either containing a monoculture or a mixed culture of protozoa and other micro-organisms, may be used in the described process.
  • the protozoa composition is dried through fluid bed drying.
  • Fluidized bed technology in dryers increases efficiency by allowing for the entire surface of the drying material to be suspended and therefore exposed to the air.
  • fluid bed drying is performed along the following method.
  • Moist material is fed onto a shaking perforated bed through which the drying air flows.
  • the air is of sufficient volume that it lifts, or 'fluidises', the bed of material allowing intimate contact with each particle.
  • the shaking action of the bed assists in the transportation of the material over the length of the dryer.
  • Moisture is carried away by the air into a dust recovery system, whereby the hot air can be recycled in a closed loop back to the process.
  • the flow of air is controlled along the length of the dryer to maximize fluidization, enabling very wet and sticky materials to be handled. As the material passes along the dryer it gradually loses moisture until the target dryness is achieved, at which point it passes into a cooling zone.
  • the hot air is replaced by cool ambient air, which reduces the product temperature to the desired figure.
  • This technology is extremely suitable for the preparation of protozoa compositions from compost tea.
  • the composition is sieved to remove large debris.
  • the sieve should have a mesh size that permits passing of micro-organisms, but retains particles larger than 0.25 mm
  • the solution is filtered through a filter with a pore size that allows passage of bacteria and archaea, but which does not allow passage of other micro-organisms (protozoa, yeasts, fungi).
  • the pore size of the filter is about 1 - 20 mu, preferably about 2 mu.
  • the retentate fraction with the protozoa, which remains on top of the filter is scraped off and can be dried on a fluid bed filter.
  • liquid protozoa compositions can also be absorbed onto a carrier material before fluid bed drying.
  • suitable carrier materials include vermiculite, perlite, diatomite, pumice, gravel, peat, fibers, such as wood, bark, coco and hemp fibers, rice husks, brick shards.
  • Drying in a fluidized bed takes place at elevated temperatures.
  • the temperature of the inlet air preferably in the range of 10 - 100°C , more preferable between 20 - 60°C .
  • Another possibility is drying on a belt dryer, which has the advantage that it can be carried out at relatively low temperatures.
  • Fytaforce PlantTM (Soiltech, Biezenmortel, The Netherlands) was spray dried in a Buchi B-290 spray drier where hot air was blown in a rate of 35,000 L/hr whereby the inlet temperature was set at various working points.
  • the Fytaforce solution was pumped from a magnetically stirred bottle through the nozzle of the spray drier.
  • a second feed liquid optionally comprising a desiccation protectant and/or further excipients were added with a second pump from a 10% (w/w dry matter) solution
  • the survival rates of protozoa were determined by direct cell counts by microscopic observation using a counting chamber.
  • Cell counts represent the total number of protozoa including flagelletes, ciliates and amoebae. To this extent the dried protozoa composition was rehydrated in Page's Amoeba Saline and incubated at room temperature for a period of 5 days. Samples were taken on a daily basis and cell counts were performed by direct microscopic observation and the results were averaged. These cell counts were compared to identical cell counts obtained from the original Fytaforce PlantTM that was spray-dried.
  • Fytaforce PlantTM (Soiltech, Biezenmortel, The Netherlands) was fluid bed dried using a laboratory scale fluid bed dryer (P.R.L. Engineering LTD). A total of 500 niL of Fytaforce Plant was dried onto 125 grams of vermiculite in 4 separate stages. At each stage 125 ml Fytaforce PlantTM was absorbed onto the 125 grams of vermiculite and dried at 30 °C at flow setting 8. At each stage samples were dried to a dry weight of > 95% w/w. The drying time to achieve this increased each stage, from 35 minutes for the first stage to 45, 60 and 70 minutes for each consecutive stage. Dried samples were vacuum sealed and stored at room temperature.
  • the survival rates of protozoa were determined by direct cell counts by microscopic observation using a counting chamber.
  • Cell counts represent the total of number protozoa including flagelletes, ciliates and amoebae.
  • the dried protozoa composition was rehydrated in Page's Amoeba Saline (PAS) and incubated at room temperature for a period of 5 days. Samples were taken after one, two and five days and cell counts were performed by direct microscopic observation and the results were averaged. These cell counts were compared to identical cell counts obtained from the original Fytaforce PlantTM that was spray-dried.
  • PAS Page's Amoeba Saline
  • PGM quality filtered data were exported in FastQ format and automatically trimmed using default parameter under ION server. Reads were de -multiplexed, barcodes and primers trimmed off and reads quality filtered under QIIME (http://qiime.org). Read containing ambiguous 'N' or shorter than 130 bps were also removed. Potential PCR chimeras were filtered out using usearch. Reads were clustered against SILVA reference euks_only database (release 111) at a sequence similarity cut-off of 97%, both closed and ope 10 strategy were used in this analysis. Aligned sequences that did not align in the appropriate zone were removed.
  • Ochrophyta (Stramenopiles) Synurophyceae, Xanthophyceae, Eustigmatophyceae

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

La présente invention concerne un procédé applicable à des processus d'obtention à l'échelle industrielle, d'une composition déshydratée de protozoaires présentant une excellente capacité de survie des micro-organismes. Cette invention est particulièrement utile pour préparer des compositions déshydratées de protozoaires à partir de thé de compost.
PCT/NL2016/050874 2015-12-16 2016-12-15 Compositions déshydratées de protozoaires et procédés d'obtention de celles-ci WO2017105231A1 (fr)

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NL2015964A NL2015964B1 (en) 2015-12-16 2015-12-16 Dried protozoa compositions.
NL2015964 2015-12-16

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997036996A2 (fr) * 1996-03-28 1997-10-09 Gist-Brocades B.V. Procede pour la preparation d'une biomasse microbienne granulaire et isolation de composes interessants a partir de cette derniere
WO2001005941A2 (fr) * 1999-07-16 2001-01-25 Public Health Laboratory Service Board Conservation de micro-organismes, de cellules et de tissus
WO2011156109A1 (fr) * 2010-06-09 2011-12-15 The United States Of America, As Represented By The Secretary Of Agriculture Procédé destiné à l'encapsulation de microparticules
WO2014043604A1 (fr) * 2012-09-17 2014-03-20 Gage Daniel Joseph Porteurs microbiens pour la répartition ciblée de charges agricoles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997036996A2 (fr) * 1996-03-28 1997-10-09 Gist-Brocades B.V. Procede pour la preparation d'une biomasse microbienne granulaire et isolation de composes interessants a partir de cette derniere
WO2001005941A2 (fr) * 1999-07-16 2001-01-25 Public Health Laboratory Service Board Conservation de micro-organismes, de cellules et de tissus
WO2011156109A1 (fr) * 2010-06-09 2011-12-15 The United States Of America, As Represented By The Secretary Of Agriculture Procédé destiné à l'encapsulation de microparticules
WO2014043604A1 (fr) * 2012-09-17 2014-03-20 Gage Daniel Joseph Porteurs microbiens pour la répartition ciblée de charges agricoles

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ARUN S MUJUMDAR: "An overview of innovation in industrial drying: current status and R&D needs", TRANSPORT IN POROUS MEDIA, KLUWER ACADEMIC PUBLISHERS, DO, vol. 66, no. 1-2, 30 August 2006 (2006-08-30), pages 3 - 18, XP019484840, ISSN: 1573-1634 *
MORGAN C A ET AL: "Preservation of micro-organisms by drying; A review", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 66, no. 2, 1 August 2006 (2006-08-01), pages 183 - 193, XP027926942, ISSN: 0167-7012, [retrieved on 20060801] *
N.N: "FYTAFORCE PLANT", 3 July 2015 (2015-07-03), Biezenmortel, pages 1 - 2, XP055281525, Retrieved from the Internet <URL:http://www.soiltech.nl/media/Productsheets_ENG/Fytaforce_Plant_1506EA.pdf> [retrieved on 20160617] *

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