NL2011862C2 - MICROBIAL LINEAR POLYSACCHARIDES. - Google Patents

Abstract

The present invention concerns a method of producing linear polysaccharides, bacteria capable of producing such linear polysaccharides, and uses of such linear polysaccharides, as a medicament, in agriculture, in food processing, as a coating, as a thickening agent, as a flocculating agent, and for water processing.

Description

Title Microbial linear polysaccharides

FIELD OF THE INVENTION

The present invention is in the field of a method of producing linear polysaccharides, bacteria capable of producing such linear polysaccharides, and uses of such linear polysaccharides, in particular chitosan, as a medicament, in agriculture, in food processing, as a coating, as a thickening agent, as a flocculating agent, and for water processing.

BACKGROUND OF THE INVENTION

The present invention relates to a method of producing linear polysaccharides, such as chitosan. Polysaccharides are long chain carbohydrate molecules having repeating monosaccharide units that are joined together by glycosidic bonds. Polysaccharides range in structure from linear to highly branched. Polysaccharides may be heterogeneous, called heteropolysaccharides or heteroglycans, containing slight modifications and/or changes in monosaccharides of the repeating unit. When all the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a homopolysaccharide or homoglycan. Depending on the structure characteristic of the polysaccharides may vary.

The term "linear" indicates that the polysaccharide is substantially linear, possibly having a few side groups, typically less than 5 wt. %, more typically less than 1 or 2 . side groups.

Chitosan is composed of randomly distributed β—(1— 4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is made by treating shrimp and other crustacean shells with the alkali sodium hydroxide.

Chitosan has a number of commercial and possible biomedical uses. It can be used in agriculture as a seed treatment and biopesticide, helping plants to fight off fungal infections. In winemaking it can be used as a fining agent, also helping to prevent spoilage. In industry, it can be used in a self-healing polyurethane paint coating. In medicine, it may be useful in bandages to reduce bleeding and as an antibacterial agent; it can also be used to help deliver drugs through the skin.

More controversially, chitosan has been asserted to have use in limiting fat absorption, which would make it useful for dieting, but there is evidence against this.

Other uses of chitosan that have been researched include use as a soluble dietary fiber. A prior art commercial process is based on extraction from an exoskeleton of crustaceans, such as crabs and shrimps, and cell walls of fungi and is inherently very polluting, toxic, relies on availability of crab and shrimp and puts pressure on the ecosystem. In' the process a (partial) deacetylation of the chitin is needed. Chitin is a precursor for chitosan in the shells. Production of chitosan by accumulating in a bulk liquid was proposed, but not being successful relating e.g. to viscosity problems, making it e.g. virtually impossible to produce at industrial scale and separate at reasonable costs. Also expensive substrates as raw material such as citrate are suggested. Typically providing citrate, or likewise glucose and the like is not sufficient for achieving any yield; in this respect other, relatively expensive, nutrients have to be provided, such as a nitrogen source.Relating to fungi, a culture medium and relatively clean, axenic and pure environmental conditions have to be provided, which makes such a process very sensitive to not well controllable parameters. Further, reactor design of prior art processes is inefficient, in terms of starting up, operation, use of chemicals, investment and operating costs.

The present invention relates to a method of producing linear polysaccharides which overcomes one or more of the above disadvantages, without jeopardizing functionality and advantages .

SUMMARY OF THE INVENTION

The present invention relates to a microbial process of producing a linear polysaccharide in dense aggregates, to an isolated culture of Defluvicoccus Vanus, to a use of Defluvicoccus Vanus in the production of a linear polysaccharide, and to a use of a linear polysaccharide, such as a polyglucosamine and β-glucosan, obtainable by a method accord- ing to the invention in various aspects.

The present method relates to cyclic reactor set up wherein a non-axenic bacteria culture is fed with a suitable carbon sources, in an aqueous environment. Therein dense aggregates of microorganisms are formed, typically in or embedded in an extracellular matrix. Such may relate to granules, to sphere like entities having a higher viscosity than water, globules, a biofilm, etc.

The present cyclic reactor supports the present cyclic process, in that a (re) circulation of reactor constituents takes place, e.g. form a first reactor/first phase to a second reactor/second phase and from a second reactor/second phase to a first reactor/first phase. As a consequence the microorganisms (and biomass) undergo cyclic conditions and/or time varying conditions.

The reactor is operated under suitable conditions e.g. for generating and growing granular sludge; the first and second stage below relate to one example.

At present, the sludge produced from e.g. wastewater treatment processes, including granular sludge, is considered as a waste product, having no further use. On top of that, costs of waste disposal are 500-600 € per ton of sludge in the Netherlands. This represents roughly one third of the wastewater treatment costs.

Surprisingly, it has been found that extracellular polymeric substances, in particular linear polysaccharides, obtainable from granular sludge can be produced in large quantities. The present invention therefore provides a commercially and environmentally very interesting application of this 'waste' product.

Granules making up granular sludge are aggregates of microbial cells self-immobilized through extracellular polymeric substances into a spherical form without any involvement of carrier material. A characterising feature of granules of granular sludge is that they do not significantly coagulate during settling (i.e. in a reactor under reduced hydrodynamic shear). Extracellular polymeric substances make up a significant proportion of the total mass of the gran- ules .

Extracellular polymeric substances comprise high-molecular weight compounds (typically >5 kDa) secreted by microorganisms into their environment. Extracellular polymeric substances are mostly composed of the present linear polysaccharides and proteins, but include other macro-molecules such as DNA, lipids and humic substances.

In general extracellular polymeric substances obtainable from granular sludge (preferably obtained from granular sludge) do not require further purification or treatment. Wherein the extracellular polymeric substances are obtained from granular sludge the extracellular polymeric substances are preferably isolated from bacteria (cells) and/or other non-extracellular polymeric substances.

Advantageously, granules of granular sludge can be readily removed from a reactor by e.g. physical separation, settling, centrifugation, cyclonic separation, decantation, filtration, or sieving to provide extracellular polymeric substances in a small volume. Compared to separating material from a liquid phase of the reactor this means that neither huge volumes of organic nor other solvents (for extraction), nor large amounts of energy (to evaporate the liquid) are required for isolation of the extracellular polymeric substances .

With the term "microbial process" here a microbiological conversion is meant. In biology, axenic relates to a culture of an organism that is entirely free of all other "contaminating" organisms. The earliest axenic cultures were of bacteria or unicellular eukaryotes, but axenic cultures of many multicellular organisms are also possible. On the contrary, the present invention makes use of a non-defined culture comprising in principle a multitude of types of microorganisms. The present non-axenic culture can in principle be taken from any soil sample, any natural water body sample, any biomass sample, etc. In principle the non-axenic culture is present in the present aqueous solution per se. In an example a non-axenic culture may be added thereto, optionally in addition to a culture already being present. By introduc ing a non-axenic microbial culture, which is considered atypical in the field of e.g. industrial large scale production processes, in principle all kinds of microorganisms may be present including ones that are capable of bioprocessing one or both types of compounds, albeit most likely in small amounts. However, initially, the culture is far from optimised, e.g. in terms of bioprocessing biomass being present. . In a way the present invention provides a method of selecting one or more microorganisms being best adapted to circumstances provided and at the same time being most beneficial for establishing desired process characteristics. Such is one of the goals of the present invention.

The present invention relates to a direct microbial production process of a linear polysaccharide, such as a po-lyglucosamide, such as chitosan in dense aggregates. The linear polysaccharide is preferably non-ionic or cationic. Inventors found a method to produce biomass, by growth of bacteria in aggregates, containing chitosan comprising a linear polysaccharide with a high content (>10 mass %). In an example chitosan is extracted and commercially used. The invention is based on operating a cyclic reactor fed in a non-aerated first stage with organic carbon containing water and, when the biomass has accumulated the carbon inside the cells thereof the reactor is in a second stage aerated. Therein the bacteria grow in dense aggregate form and are the end of a cycle maintained in the reactor e.g. by settling or another physical separation process.

Inventors now can directly produce chitosan, without a need of deacetylation. The present reactor is run under non-axenic conditions, having advantages over the prior art such as investment costs that are significantly lower. The process can run on relatively cheap and readily available substrates, such as biomass, acetate and propionate. Also waste organic carbon and products that can be obtained easily from waste organic carbon are used.

In an example chitosan is a polymer that is produced by bacteria, in particular candidatus Defluvicoccus Vanus.

Such is further substantiated by presence of D. Vanus DNA in samples. It is noted that currently there is no microbial chitosan production process.

It is considered rather unexpected that bacteria can produce a linear polysaccharide such as chitosan. Even further surprisingly. a linear polysaccharide such as chitosan can be produced in an enriched microbial community e.g. growing on waste organic carbon. Until now the general perception was that anionic alginate is the main gel forming agent in microbial aggregate growth systems.

The present invention provides a method that amongst others has less environmental pollution. There is no need for deacetylation. The high prior art cost price of chitosan (10 Euro/kg) due to a limited market supply from these sources, is now overcome. Currently the supply is seasonal depended, which is no longer the case with the present invention. Further, production in controlled reactor conditions gives a more stable quality of polymer. Also in terms of substrate costs and technical feasibility the present method is a major step forwards.

Inventors now can produce linear polysaccharides from waste thereby contributing strongly to a biobased or circular economy. Due to a decrease in price the cationic polyelectrolyte may find much more applications. The present linear polysaccharide, such as chitosan, is considered a green chemical in the sense that it is derived from natural resources and is fully biodegradable when introduced in a natural environment.

Thereby the present invention provides a solution to one or more of the above mentioned problems.

Advantages of the present description are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to a microbial process of producing a linear polysaccharide in dense aggregates. Therein a non-axenic bacteria culture, a suitable carbon source, and water are fed to a cyclic reactor. The carbon source may be one or more of an organic car bon source, such as acetate, propionate, biomass and waste organic carbon. The waste organic carbon source may be a waste organic sludge source. In an example the present invention relates to a process wherein the aqueous carbon substrate-comprising solution is wastewater originating from one or more of domestic residences, commercial properties, industry, and agriculture, such as sewage, paper production waste-water, solid organic waste processing/leaching water, and food production wastewater. In a further example the aqueous carbon substrate-comprising solution is limited in phosphorous concentration, (substantially) preventing poly-P formation .

In general terms, the present process can be implemented in a continuous mode, semi-continuous mode, and in a sequenced batch mode. Throughout the description the term "reactor" may also relate to a "phase", and vice versa, if applicable. In an example, the proposed system is composed of two types of reactors or phases.

In a first stage under low oxygen concentration conditions (anaerobic) during a predetermined period of time at a predetermined temperature accumulating carbon in cells of the bacteria culture. The oxygen concentration is preferably as low as possible, e.g. with respect to a reactor set-up.

The period of time is in the order of 0.1-8 hours, preferably 0.25-4 hours, more preferably 0.5-2 hours, such as 1 hour.

The times are typically calculated/determined as solid retention time. The temperature is preferably maintained at a predetermined temperature of 1 °C - 50 °C, preferably 30 °C - 40 °C.

In the first stage the solution may comprise one or more nutrients selected from an N-source, such as ammonia, an Mg-source, a P-source, an S-source, a K-source, and spore elements - such as 50-100 mM NH4+, 10-40 mM K+, 2-10 mM Mg+, 2-10 mM S042- - and/or a nitrification inhibitor, such as al-lythiourea, such as 50-200 mg/1 allythiourea. It is noted that albeit in principle addition of nutrients may support growth of bacteria and might be considered, such is for the present process not needed. In the first stage it is pre ferred to have a phosphorous concentration relatively low; the phosphorous concentration is high enough to support cell construction, but low enough to (substantially) prevent forming of polyphosphate (s) .

In a second stage the bacteria are grown in aggregate form under relatively high (elevated) oxygen concentration conditions (aerobic) during a predetermined period of time at a predetermined temperature growing. The oxygen concentration may be similar or equal to environmental conditions. It is noted that an oxygen concentration of 10% of a saturation value is considered high enough in this respect. The second stage time is 3-48 hours, preferably 6-24 hours, more preferably 10-18' hours, such as 12 hours. The temperature is preferably maintained at a predetermined temperature of 1 °C - 50 °C, preferably 30 °C - 40 °C.

As a consequence the bacteria in the aggregates grow on the cell internal stored materials and produce polysaccharides, especially chitosan under the described conditions. In the process also new aggregates are formed spontaneous leading to a net formation of aggregate biomass containing polysaccharides .

In an alternative a waste water supply further comprises an ammonium source; and wherein first and second stage are "combined" and comprise maintaining the reactor under high oxygen concentration conditions (aerobic).

It is then possible to separate the aggregates, the aggregates comprising the linear polysaccharide, such as by physical separation, settling, centrifugation, cyclonic separation, decantation, filtration, or sieving. The aggregates are retained in the reactor system and used in the next cycle while the net production of new aggregates is harvested for chitosan extraction.

In an example, the extracellular polymeric substances comprise at least 50 % w/w linear polysaccharides, preferably at least 60 % w/w linear polysaccharides, most preferably at least 75 % w/w linear polysaccharides, such as at least 90 % w/w linear polysaccharides.

In an example, the granular sludge is aerobic granu- lar sludge or anammox granular sludge. Aerobic granular sludge and anammox granular sludge, and the processes used for obtaining them are known to a person skilled in the art. For the uninitiated, reference is made to Water Research, 2007, doi:10.1016/j.watres.2007. 03. 044 (anammox granular sludge) and Water Science and Technology, 2007, 55(8-9), 75-81 (aerobic granular sludge).

In an example, the extracellular polymeric substances have been obtained from aerobic or anammox granular sludge by an isolation (i.e. separation) method comprising: alkaline extraction of the granular sludge thereby forming extracellular polymeric substances containing extractant; acid precipitation of extracellular polymeric substances from the extractant; and collecting the extracellular polymeric substance-containing precipitate .

The present process is preferably performed at a pH of 4-12, such as 6.5-8.0.

In an example a size of the first reactor used for the first stage is 0.15-6 times the size of the second reactor used for the second stage, preferably 0.5-2 times the size of the second reactor, more preferably 0.66-1.5 times the size of the second reactor, even more preferably 0.8-1.25 times the size of the second reactor, such as 0.9-1.1 times. In other words relatively small reactors can be used, providing good yields and quick throughput times.

Each reactor may comprise a temperature regulator, a pump, a mixer, and one or more controllers.

In an example the bacteria culture comprises Deflu-vicoccus Vanus (D. Vanus) bacteria and the linear polysaccharide is chitosan (polyglucosamine).

In an example the chitosan is extracted from the aggregates .

In an example the cyclic reactor comprises two stages or two reactors in mutual connection. Such is described in more detail above.

In an example a fraction of the bacteria, such as 0-100% of the bacteria in aggregate form, is fed to the first stage. As such the first stage is enriched in terms of pres ence (or concentration) of bacteria capable of forming a linear polysaccharide. Likewise, no required bacteria slowly die out. Preferably 10-98% of the bacteria in aggregate form are fed back, more preferably 80-95%. Such will depend on conditions of operation, such as a relative reactor size, and a ratio between cycle time and solid retention time.

In a second aspect the present invention relates to an isolated culture of Defluvicoccus Vanus deposited on October 28, 2013, at the Centraal Bureau voor Schimmelcultures (CBS) the Netherlands under accession number CBS 136885, a mutant strain thereof, a recombinant strain thereof, and spores thereof. It has been found that especially the D.

Vanus is very suited for the present process, e.g. in terms of yield, conversion, tolerant to feed stock, etc.

In a third aspect the present invention relates to a use of the present Defluvicoccus Vanus in the production of a linear polysaccharide, such as a polyglucosamine and β-glucosan. Details thereof are given throughout the description. It is noted that side products may be formed, albeit in small quantities.

In a fourth aspect the present invention relates to a use of a linear polysaccharide, such as a polyglucosamine and β-glucosan, obtainable by a method according to the invention, as a medicament, such as for limiting fat absorption, for dieting, for increasing excretion of sterols, for reducing digestibility of ileal fats, for inhibiting uptake of dietary lipids, as a soluble dietary fibre, in bandages for reducing bleeding, as an antibacterial agent, as a haemostatic agent, as a hypoallergenic agent, and as a vehicle to deliver drugs through the skin. As the present process provides very pure linear polysaccharides, having only minute amounts of impurities, especially after separation, the present linear polysaccharides are especially suited for use as a medicament.

In a fifth aspect the present invention relates to a use of a linear polysaccharide, such as a polyglucosamine and β-glucosan, in one or more of agriculture, such as for plant defence, for yield increase, as plant growth enhancer, for seed treatment, as biopesticide, as a biocontroller, and as a fungicide, food processing, such as a fining agent in winemaking, and in preventing spoilage, a coating, such a self-healing polyurethane (paint) coating, a drug carrier, a sheet, such as for culturing stem cells, a laminate, such as a multi laminate, a non-woven product, cosmetics, an additive, such as a thickening agent and a flocculating agent, for water processing, for forming of biobased composite materials, in combination with nanoparticles, for binding metals, for binding metal oxides such as arsenate and uranate, for binding oil, and for binding phosphorous. The present linear polysaccharide finds many applications, of which a few are mentioned above. In view of a price of the present linear polysaccharide, especially in comparison to a price of prior art products, the application will be considered more frequent .

The invention is further detailed by the accompanying examples, which are exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims .

EXAMPLES/EXPERIMENTS

As described a cyclic reactor was operated, providing in a first stage organic carbon containing water under non-aerated conditions. The biomass accumulated. Then in a second stage the reactor was aerated. The present bacteria grow in dens granular form. With the above method in an initial set up biomass was obtained with a 10-20% chitosan content (wt/chitosan/wt. total biomass, based on dry weigth) . At the end of a cycle the granules were settled in the reactor. The obtained chitosan was then extracted. The extracted chitosan may then be further purified .

Experiment 1 setup Chitosan reactor A double walled glass sequencing batch reactor (SBR) with an internal diameter of 6.25cm, 1.5m in height and 2.7 liters working volume was operated as a bubble column. Activated sludge from a conventional waste water treatment plant in the Netherlands (Harnaschpolder), that had excellent nitrogen and phosphate removal capability, was used to inoculate the reactor. The temperature of the reactor was controlled at 35+0.5°C by means of a cryostat. The influent was preheated in order to ensure that the reactor was fed with at a correct temperature. The off gas was recirculated with a constant flow of 5 L min-1 to keep the dissolved oxygen at its desired set point' of 50% (of the maximum solubility of oxygen at 35 °C) . The dissolved oxygen was controlled by two mass flow controllers for nitrogen gas and air. Normally only air was supplied to the reactor since the bacteria are using oxygen. A bio controller (Braun DCU4 coupled with Multi Fermentor Control System acquisition software; Sartorius Stedim Biotech S.A., Melsungen, Germany) was used to control and operate the sequencing batch reactor. The exchange ratio was 0.56. The reactor was operated with a cycle length of 3 hours, following an anaerobic feast-aerobic famine regime as shown in table 1. A dosage of 1M NaOH and HC1 controlled the pH at (7.1+0.05). The granular sludge used to seed the reactor was taken from a granular lab reactor in use for anaerobic methane production from methanol (Phase 1) . The total suspended solids (TSS) and volatile suspended solids (VSS) were calculated. The solid retention time (SRT) was maintained at 20-25 days.

Medium A synthetic medium consisted of 150 ml medium A and 150 mL medium B dosed together with 1200 mL heated tap water was used, achieving an influent temperature of 35°C. The composition of medium A consisted of NH4C1 21 mM, K2HP04 0.72 mM, KH2PO4 0.37 mM, MgS04-7H20 3.6 mM, KC1 4.7 mM, and 9mL/L trace element solution (M. 1957). The composition of medium B consisted of: HAc 67.6 mM, MeOH 15.6 mM.

Table 1. Cycle timing

Phases Time [min] Volume [L]

Anaerobic feeding 60 1.5

Aeration 100-112

Settling 15-3

Effluent withdrawals 1.5

Total cycle length 180

Depending on the use intended, further purification of chitosan may be required or not.

The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying examples.

It should be appreciated that for commercial application it may be preferable to use one or more variations of the present system, which would similar be to the ones disclosed in the present application and are within the spirit of the invention.

Claims (10)

A microbial method for producing a linear polysaccharide in dense aggregates, comprising the steps of (i) feeding a cyclic reactor with (a) a non-axenic bacterial culture, (b) a suitable carbon source, such as an organic carbon source, such as acetate, propionate, biomass and organic carbon waste, and (c) water (ii) in a first phase under low oxygen concentration conditions (anaerobic) for a predetermined time at a predetermined temperature accumulating carbon in cells of the bacterial culture, (iii) in a second phase providing elevated oxygen concentration conditions (aerobically) for a predetermined time at a predetermined temperature, culturing the bacteria in aggregated form, and (iv) separating the dense aggregates, wherein the dense aggregates aggregates include the linear polysaccharide, for example by physical separation, settling, centrifugation, cyclone separation, decanting, filtering or sieving. The method of claim 1, wherein the bacterial culture comprises Defluyicoccus Vanus (D. Vanus) bacteria and the linear polysaccharide is chitosan (polyglucosamine). The method of any one of claims 1-2, wherein the chitosan is extracted. The method of any one of claims 1-3, wherein the cyclic reactor comprises two stages or two reactors in interrelation. The method of any one of claims 1-4, wherein 0-100% of the bacteria are supplied in aggregate form to the first stage. The process of any one of claims 1-5, wherein a temperature of the cyclic reactor is maintained at 30-50 ° C, preferably at 35-40 ° C. 7. An isolated culture of Defluvicoccus Vanus deposited with CBS in the Netherlands under accession number CBS 136885, a mutant strain thereof, a recombinant strain thereof, and traces thereof. Use of Defluvicoccus Vanus according to claim 7 in the production of a linear polysaccharide, such as a polyglucosamine and β-glucosan. Use of a linear polysaccharide, such as a polyglucosamine and β-glucosan, obtainable by a method according to any one of claims 1-6, as a medicine, for example for limiting fat intake, as a diet, for the excretion of sterols, for the reduce digestibility of ileum fats, to inhibit the uptake of dietary lipids, as soluble dietary fibers, in dressings for reducing bleeding, as an antibacterial agent, as a hemostatic agent, as a hypoallergenic agent, and as a drug delivery agent via the skin. Use of a linear polysaccharide, such as a polyglucosamine and β-glucosan, obtainable by a method according to any one of claims 1-6, in one or more of agriculture, such as for the defense of plants, for yield enhancement, such as plant growth enhancer for seed treatment, as a biopesticide, as a biocontroller, and as a fungicide, food processing such as a clarifying agent in winemaking, and to prevent spoilage, a coating such as a self-healing polyurethane (paint) coating, a medicine carrier, a leaf, such as growing stem cells, a laminate, such as a multi-laminate, a non-woven product, cosmetics, a. additive, such as a thickener, and a flocculant, and for water treatment, for forming biobased composite materials, in combination with nanoparticles, for binding metals, for binding metal oxides such as arsenate and uranate, for binding oil, and for phosphorus binding. >