WO2007120801A2 - Fermentation method - Google Patents

Abstract

Methods of saline fermentation are provided. A fermenter vessel is charged with a fermentation medium having less than about 300 ppm chloride ions, which is then sterilized. A sterile chloride salt solution is added to the medium to produce a saline fermentation medium. The saline fermentation medium is then inoculated with a microorganism and the saline fermentation medium is cultured under conditions suitable for the growth of the microorganism. Finally, the medium can be harvested.

Description

FERMENTATION METHOD

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 60/791,625, filed April 13, 2006, and New Zealand Patent Application No. 546572, filed April 13, 2006, which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

[0002] The present invention relates to a method of saline fermentation, in particular to methods of minimizing corrosion to fermenters during saline fermentation.

Description of the Related Art

[0003] Industrial fermentation involves the large scale cultivation of microorganisms for the production of desirable compounds via metabolism or recombinant protein expression. Desirable compounds include primary metabolites such as ethanol, citric acid, dextran and xanthan gum; secondary metabolites such as antibiotics, antifungal and immunosuppressant agents, and enzymes such as cellulases, proteases and lipases. The transformation of microorganisms with expression constructs have also been used to produce a number of therapeutic proteins, such as interferon, insulin, and human serum albumin.

[0004] The discovery rate of potentially valuable compounds in microorganisms from saline environments is increasing. In recent years marine microorganisms have become important in the study of novel microbial products exhibiting antimicrobial, antiviral, antitumor as well as anticoagulant and cardioactive properties.

[0005] In many cases these microorganisms cannot be adapted to grow in low salt media and as a result must be grown in media mimicking their natural high salt environments. High salt solutions present major corrosion problems for traditional stainless steel fermenters which are rapidly pitted by chloride ions, particularly at elevated temperatures. Chloride-induced pitting corrosion is initiated at concentrations of about 500 parts per million (ppm) chloride and will greatly reduce the useful lifetime of the fermenter and result in the eventual failure of the fermenter. [0006] As a consequence, the common 300 series austenitic stainless steels are limited to holding only trace amounts of chlorides. For example, grade 304 stainless steel can be used to hold water containing 100 to 300 ppm chlorides at temperatures below 50⁰C. However, high concentrations of chlorides are required for saline fermentation, where artificial seawater conditions are necessary to replicate the growth conditions of microorganisms such as Salinospora tropica. The chloride content of seawater is approximately 18,000 ppm. High temperatures are also used to sterilize the fermentation media before and after fermentation and to sterilize the fermenter after use. In addition, the fermentation itself often requires an elevated temperature for optimal growth of the microorganism.

[0007] Approaches suggested for reducing the corrosion of stainless steel caused by saline fermentation include the use of non-chloride sodium salts, the use of ceramic or silicon coatings to provide a protective film for the stainless steel, or the use of specialized stainless steel alloys having enhanced corrosion resistance. However, the cost of such alternatives is often prohibitive and not applicable for the saline fermentation of microorganisms on an industrial scale. Moreover, use of non-chloride salts is not useful when trying to mimic the high salt marine environment.

[0008] Accordingly, it is an object of the present invention to provide a method of saline fermentation which goes at least some way towards addressing the problems disclosed above, or which at least provides the public with a useful choice.

SUMMARY OF THE INVENTION

[0009] One aspect of the invention relates to a saline fermentation method comprising:

(a) charging a fermenter vessel with a fermentation medium having less than about 300 ppm chloride ions; Qo) sterilizing the medium in the vessel;

(c) adding a sterile chloride salt solution to the medium to produce a saline fermentation medium;

(d) inoculating the saline fermentation medium with a microorganism and culturing the saline fermentation medium under conditions suitable for the growth of the microorganism; and

(e) harvesting the medium. [00101 Where the fermenter is intended to be used repeatedly, it is preferable before re-using to:

(i) inspect the vessel and remove any corrosion present; and

(ii) repassivate the vessel.

[0011] Another aspect of the invention relates to a method of minimizing corrosion of a stainless steel vessel during saline fermentation of a microorganism, comprising:

(a) charging a fermenter vessel with a fermentation medium having less than about 300 ppm chloride ions;

(b) sterilizing the medium in the vessel;

(c) adding a sterile chloride salt solution to the medium to produce a saline fermentation medium;

(d) inoculating the saline fermentation medium with a microorganism and culturing the saline fermentation medium under conditions suitable for the growth of the microorganism; and

(e) harvesting the medium.

[0012] Where the fermenter is intended to be used repeatedly, it is preferable before re-using to:

(i) inspect the vessel and remove any corrosion present; and

(ii) repassivate the vessel.

[0013] In one embodiment the fermentation medium in the fermenter vessel is heat sterilized.

[0014] Heat sterilization may be carried out between about 75°C and about 140⁰C for 15 minutes or more. Preferably, the fermentation medium is heat sterilized at about 121°C for about 45 minutes.

[0015] The fermenter vessel is one constructed of a material subject to corrosion at high salt concentrations. In preferred embodiments the fermenter vessel is constructed of stainless steel. Stainless steels contemplated for use in the invention include 300 series austenitic stainless steels, preferably 304, 304L, 316 or 316L grade stainless steels.

[0016] In other embodiments where the fermentation requires more severe chloride concentrations and/or sustained elevated temperatures, more resistant stainless steel grades having higher levels of molybdenum, chromium, and nitrogen additions may be used. Such grades include the "super austenitic" grades (for example N08904 and S31254) and the "super duplex" grades (for example S32750 and S32520).

[0017] The stainless steel fermenter preferably has a Pitting Resistance Equivalent (PRE) number less than 45, preferably less than 40.

[0018] In one embodiment the saline fermentation medium has a chloride ion concentration between about 6,500 ppm and about 240,000 ppm. Currently preferred saline fermentation media have a range of chloride ion concentration between about 6,500 ppm and about 90,000 ppm, preferably about 11 ,000 ppm and about 60,000 ppm, more preferably about 12,000 ppm and about 50,000 ppm, more preferably about 13,000 ppm and about 40,000 ppm, more preferably about 14,000 ppm and about 30,000 ppm, most preferably about 15,000 ppm and about 20,000 ppm.

[0019] In one preferred embodiment the saline fermentation medium has a chloride ion concentration of about 18,000 ppm.

[0020] The chloride salt solution may comprise one or more chloride salts. In one embodiment the chloride salt solution comprises one or more chloride salts selected from calcium chloride (CaCl₂), cobalt chloride (CoCl₂), cesium chloride (CsCl), potassium chloride (KCl), lithium chloride (LiCl), magnesium chloride (MgCl₂), manganese chloride (MnCl₂), sodium chloride (NaCl), ammonium chloride (NH4CI), nickel chloride (NiCl₂), rubidium chloride (RbCl), zinc chloride (ZnCl₂), sodium perchlorate (NaClO₄), and ferric chloride (FeCb).

[0021] Currently preferred for use is a chloride salt solution comprising sodium chloride (NaCl).

[0022] In one embodiment the sodium chloride concentration in the saline fermentation medium is between about 11 g/L and about 400 g/L. Most usually, the sodium chloride concentration in the saline fermentation medium is between about 11 g/L and about 150 g/L, preferably about 18 g/L and about 100 g/L, more preferably about 20 g/L and about 83 g/L, more preferably about 21.5 g/L and about 67 g/L, more preferably about 23 g/L and about 50 g/L, most preferably about 24 g/L and about 33 g/L.

[0023] In one preferred embodiment the sodium chloride concentration in the saline fermentation medium is about 30.2 g/L.

[0024] The microorganism to be fermented may be any microorganism capable of being grown under saline fermentation conditions and includes bacteria, protozoa, algae, cyanobacteria, yeast and fungi. [0025] Preferably, the microorganism is a halotolerant or halophilic microorganism.

[0026] In one embodiment the selected microorganism is a marine microorganism.

[0027] Microorganisms amenable to saline fermentation include Actinopolyspora, Haloferax, Halobacterium, Halobacillus, Marinilactibacillus, Spirochaeta, Vibrio, Pseudomonas, Haloarcula, Rhodothermus, Halomonas, Halococcus, Salinospora, Anaerophaga, Haloanaeroba, Debaryomyces, Scytalidium, Hyphomycetes, Zygomycetes, Ascomycetes, Trimmatostroma, and Hortaea.

[0028] In one embodiment the microorganism is Salinospora tropica or Pseudoalteromonas antarctica.

[0029] In one embodiment the fermentation is maintained at a temperature between about 10⁰C and about 70⁰C, preferably about 20⁰C and about 65°C, preferably about 25°C and about 50⁰C, more preferably about 26°C and 40⁰C, most preferably about 28°C.

[0030] In one embodiment the pH of the saline fermentation medium is maintained between about pH 1 to about pH 11.

[0031] In one embodiment the pressure of the saline fermentation is maintained between about 1 to about 100 bar.

[0032] The saline fermentation medium is desirably kept in motion during the culture. This may be achieved for example through mechanical mixing and/or gas- sparging.

[0033] In one embodiment the fermentation is aerobic.

[0034] In an alternative embodiment the fermentation is anaerobic.

[0035] The saline fermentation medium may be inactivated after the culture step is completed, either before or after harvesting the medium from the fermenter vessel.

[0036] Inactivation may be effected in a variety of ways including in situ by heating or by addition of a chemical biocide.

[0037] In one embodiment the saline fermentation medium is harvested and inactivated in a separate process vessel. A useful process vessel is a heat exchanger.

[0038] The corrosion in the fermenter may comprise pitting corrosion, crevice corrosion or stress corrosion cracking. [0039] In one embodiment the fermenter vessel is visually inspected for evidence of corrosion after fermentation.

[0040] Where the fermenter vessel is to be reused any corrosion present after fermentation may be removed by mechanical abrasion and polishing, using varying grades of abrasive paper or abrasive pads.

[0041] In one embodiment the vessel is repassivated following fermentation using known art techniques. Nitric acid passivation is currently preferred.

[0042] In another aspect the present invention relates to microorganisms and compounds therefrom produced by the methods of the invention.

[0043] All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0044] This invention may also be broadly said to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of the parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Further aspects and advantages of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings, wherein,

[0046] Figure 1 shows pitting potentials of grades 304, 316 and 904L stainless steels in 1 M NaCl solution, with a 220 grit surface finish, as a function of temperature.

[0047] Figure 2 shows the pitting potential, Epj_t, measured for grades 302 (similar to grade 304) and 316 stainless steels in various NaCl solutions, with a 120 grit surface finish.

[0048] Figure 3 shows the pitting potentials of grade 316L stainless steel in artificial seawater (Instant Ocean®, Aquarium Systems, Ohio USA), with and without the addition of bacterial growth medium. The two curves on the right of the plot represent 28°C, while the two curves on the left of the plot represent 50⁰C. Diamonds represent Instant Ocean® solution and the squares represent Instant Ocean® + media solution.

DETAILED DESCRIPTION OF THE INVENTION

[0049] Saline solutions are known to rapidly corrode stainless steel, particularly at elevated temperatures. Stainless steel vessels are therefore generally regarded as not suitable for saline fermentation methods, as corrosion greatly reduces the useful lifetime of the fermenter resulting in the eventual failure of the vessel.

[0050] Unexpectedly, the present inventors have shown that saline fermentation can be routinely conducted in stainless steel vessels if certain steps are taken to minimize corrosion.

[0051] The present invention is therefore directed to methods of minimizing corrosion caused by saline solutions during saline fermentation.

[0052] The term "fermentation" as used herein refers to a process for the production of one of more desired compounds formed by the aerobic or anaerobic metabolism of the microorganism in a medium suitable for the growth of the microorganism.

[0053] The term "saline fermentation" as used herein refers to a fermentation process conducted with a medium having a chloride concentration above about 6,500 ppm

[0054] The term "fermentation medium" as used herein refers to a medium comprising the ingredients necessary required for microorganism growth and production of the desired compound. The medium will usually include carbon, nitrogen, phosphorus and sulphur sources, nutrients, trace elements, salts vitamins and so forth. The varying fermentation requirements for a wide variety of microorganisms are well known in the art.

[0055] The term "saline fermentation medium" as used herein refers to a fermentation medium having a chloride concentration above about 6,500 ppm.

[0056] The term "under conditions suitable" as used herein refers to the physical and chemical parameters of the fermentation medium necessary for the growth of the microorganism and/or production of the desired compound, comprising pH, temperature, salinity, pressure, dissolved oxygen concentration, nitrogen requirements and substrate, nutrient and trace element concentrations and the like. [0057] The term "repassivating" as used herein should be taken to mean a reduction of the anodic reaction rate of a metal surface subject to corrosion by changing the chemically active surface of the metal to a less reactive state. Passivation can be effected by any known art techniques including chemically treating the metal surface with a mild oxidant such as nitric acid solution to enhance the spontaneous formation of a protective passive film.

[0058] The term "pitting corrosion" as used herein should be taken to mean the localized corrosion of a metal surface in the form of cavities or pits. Pitting occurs mainly in the presence of neutral or acidic solutions containing chlorides which facilitate a local breakdown of the passive surface to create an active cavity or pit that rapidly corrodes.

[0059] The term "crevice corrosion" as used herein should be taken to mean corrosion that occurs in a crevice that is of sufficient width to permit entry of a corrodent, but sufficiently narrow to ensure that the corrodent remains stagnant. Under such localized conditions the protective oxide layer of stainless steel breaks down, resulting in severe, localized corrosion within the crevice region.

[0060] The term "stress corrosion cracking" (SCC) as used herein should be taken to mean the brittle cracking of a metal under tensile stress, enhanced by the presence of a corrodent. For austenitic stainless steels, the most common type is transgranular chloride-induced SCC, that may develop in concentrated chloride- containing environments at temperatures above 50 ⁰C and which typically takes the form of thin, branched cracks.

[0061] The term "comprising" as used herein means "consisting at least in part of. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present.

[0062] Protocols for the fermentation of microorganisms are well known in the art. Fermentations on an industrial scale are typically carried out in large fermenter vessels containing a fermentation medium comprising the necessary ingredients for microorganism growth. The medium is first heat sterilized to remove any contaminating microorganisms, before being inoculated with the microorganism to be fermented and incubated under controlled conditions for one to two weeks. [0063] Following incubation the medium is once again heated to kill the microorganisms and the medium harvested for processing and isolation of the desired compounds.

[0064] In preferred embodiments the fermenter vessels are constructed of stainless steel, which offers corrosion resistance, ease of cleaning, and ability to easily maintain temperature control.

[0065] Over 70 different types of stainless steel are available. Due to their formability and corrosion resistance, austenitic stainless steels of the 200 and 300 series having 16 to 30% chromium and 2 to 20% nickel are preferably used.

[0066] In some embodiments the fermenter vessels are constructed from grade 304 (having a PRE number of 19) or grade 316 (having a PRE number of 25) stainless steel.

[0067] In other embodiments stainless steel alloys such as the "super austenitic" grades (for example N08904 and S31254) containing up to 6% molybdenum and the "super duplex" grades (for example S32750 and S32520) having high chromium, molybdenum and nitrogen additions may be used to extend the resistance of stainless steel in high chloride environments.

[0068] The method of the invention comprises charging a fermenter vessel with a fermentation medium having less than about 300 ppm chloride ions and heat sterilizing the medium to eliminate contaminating microorganisms.

[0069] Heat sterilization may be carried out between about 75°C and about 140⁰C for 15 minutes or more.

[0070] In preferred embodiments the medium is heat sterilized by heating the medium to about 121°C, holding the medium at that temperature for about 45 minutes, then cooling the medium to the desired culture temperature.

[0071] To minimize the time the chloride salt solution is in contact with the stainless steel at elevated temperatures, a high concentration sterile chloride salt solution is added to the fermentation medium subsequent to the initial sterilization step and after cooling to the desirable temperature, to produce a saline fermentation medium.

[0072] In preferred embodiments the high concentration chloride salt solution is added to the fermentation medium via a sterile filter, preferably a filter with a pore size of about 0.2 microns or less.

[0073] In other embodiments the high concentration chloride salt solution is sterilized by known methods, prior to addition to the fermentation medium. [0074] A microorganism culture is then added to the fermenter vessel to start the fermentation process.

[0075] In one embodiment the microorganism is first added to a seed vessel containing previously sterilized saline fermentation medium under conditions suitable for growth of the microorganism, allowing previously frozen or lyophilized cells to activate. Following the seed fermentation the culture is added to the fermenter vessel.

[0076] In one embodiment the microorganism is a bacterium, protozoan, alga, cyanobacterium, yeast or fungus, preferably a halotolerant or halophilic microorganism.

[0077] The discovery rate of potentially valuable compounds in microorganisms from saline environments is increasing, with a large collection of microorganisms already described in the literature.

[0078] A collection and screening process may also be used to isolate new strains of microorganisms. Suitable environments from which to select microorganisms include shallow, saline habitats which preferably undergo a wide range of temperature and salinity variation such as marine environments, tide pools, estuaries and inland saline ponds and lakes and thermal springs.

[0079] In preferred embodiments the selected microorganism is a marine microorganism.

[0080] Following inoculation the saline fermentation medium is cultured under conditions suitable for the growth of the microorganism and production of the desired compound.

[0081] The parameters of the fermentation conditions, such as pH, temperature, salinity, pressure, dissolved oxygen concentration, carbon, nitrogen, phosphorus and sulphur requirements, nutrient and trace element concentrations may be varied to meet the requirements of the microorganism being fermented and the conditions required for production of the desired compound.

[0082] The conditions for the growth of the microorganism may vary from the conditions necessary for the production of the desired compound. In such embodiments the conditions can be altered once the microorganism reaches the end of the growth phase.

[0083] For example, halophilic microorganisms are generally multiplied in an aerobic culture. In contrast, the production of the desired compound may be induced in an oxygen-depleted or anaerobic culture. Similarly, other conditions may be altered to induce production of the desired compound. [0084] In some embodiments the entire fermentation process may be carried out in a partially anaerobic range so that growth and induction occur concurrently.

[0085] In other embodiments the entire fermentation process may be carried out aerobically, with production of the desired compound being induced by altering one or more of the other culture conditions.

[0086] In an anaerobic culture the fermentation is carried out in an oxygen- free medium or with small amounts of up to about 3 vol. %, preferably about 2.5 vol. %, more preferably about 2 vol. % and most preferably about 1.5 vol. % oxygen.

[0087] In a partially anaerobic range culture the fermentation is carried out with oxygen contents of about 3 and about 8 vol. %. The oxygen content is preferably between about 3.5 to about 5 vol. %.

[0088] In an aerobic culture the fermentation is carried out between about 8 vol. % to about 23 vol. % oxygen, preferably between about 10 vol. % and about 23 vol. % and more preferably between about 15 vol. % oxygen and about 23 vol. %. When reference is made to oxygen content, it relates to the oxygen content in the gas mixture that is fed into the reaction medium or to the oxygen content above the reaction medium. The oxygen content in the gas phase can be measured by conventional methods e.g. by means of oxygen electrodes.

[0089] The amount of oxygen dissolved in the saline fermentation medium is dependent on the amount of oxygen supplied or on the amount of oxygen present in the gas phase, whereby an equilibrium is established between the oxygen dissolved in the saline fermentation medium and the oxygen present in the gas phase. In the methods of the invention oxygen may be passed into the saline fermentation medium using air sparging, or into the gas phase which then accordingly dissolves in the saline fermentation medium. The dissolved oxygen levels can be monitored and oxygen added as required.

[0090] The solubility of oxygen in the saline fermentation medium is dependent on temperature.

[0091] Preferably the fermentation is maintained at a temperature between about 10⁰C and about 70 ⁰C, preferably about 20⁰C and about 65°C, preferably about 25°C and about 50⁰C, more preferably about 26°C and 40⁰C and most preferably about 28°C. (0092] The saline fermentation medium preferably has a sodium chloride concentration about 11 g/L by weight up to the content of a saturated solution of about 400 g/L. At 25°C a saturated sodium chloride solution contains about 260 g/L. A sodium chloride solution containing about 30 g/L by weight corresponds approximately to the sodium chloride concentration of sea-water.

[0093] In one embodiment the sodium chloride concentration in the saline fermentation medium is between about 11 g/L and about 150 g/L, preferably between about 18 g/L and about 100 g/L, more preferably about 20 g/L and about 83 g/L, more preferably about 21.5 g/L and about 67 g/L, more preferably about 23 g/L and about 50 g/L, most preferably about 24 g/L and about 33 g/L.

[0094] In one preferred embodiment the sodium chloride concentration in the saline fermentation medium is about 30.2 g/L.

[0095] Halophilic microorganisms grow optimally Ln media containing a salt concentration of about 30 g/L. Extremely halophilic microorganisms and halotolerant microorganisms grow optimally at salt concentrations of about 150 g/L. Examples of extremely halophilic organisms include Haloferax volcanii and Halobacterium salinarum.

[0096] Extremely halophilic microorganisms are viable at concentrations of about 100 g/L.

[0097] Halotolerant microorganisms can grow in high concentration salt solutions of about 150 g/L, compensating the osmotic pressure by producing a suitable substance such as glycerol. Such organisms are viable at low as well as at very high salt concentrations.

[0098] In one embodiment the saline fermentation medium has a chloride ion concentration between about 6,500 ppm and about 240,000 ppm. Currently preferred saline fermentation media have a range of chloride ion concentration between about 6,500 ppm and about 90,000 ppm, preferably about 11 ,000 ppm and about 60,000 ppm, more preferably about 12,000 ppm and about 50,000 ppm, more preferably about 13,000 ppm and about 40,000 ppm, more preferably about 14,000 ppm and about 30,000 ppm, most preferably about 15,000 ppm and about 20,000 ppm.

[0099] In one preferred embodiment the saline fermentation medium has a chloride ion concentration of about 18,000 ppm.

[0100] The pH of the saline fermentation medium may vary between about pH 1 and about pH 11. [0101] The pressure under which the saline fermentation is maintained will also preferably be chosen to match the desired conditions for growth of the microorganism and production of the desired compound, and may vary between about 1 bar and about 100 bar.

[0102] To minimize stagnant areas in the saline fermentation medium which will lead to biofilm formation and an acidic and reducing environment, the saline fermentation medium must be kept in motion to prevent corrosion.

[0103] In preferred embodiments the saline fermentation medium is kept in motion during the culture, preferably through mechanical mixing and/or gas-sparging.

[0104] The desirable compounds produced by microorganism fermentation include primary metabolites produced during the log or exponential phase of microorganism cell growth (tropophase) that are essential for the cell's growth, such as enzymes, nucleotides, nucleic acids, amino acids, proteins, carbohydrates and lipids; or by-products of metabolism such as ethanol, acetone and butanol. For example, Antarticine-NF3, a glycoprotein produced by Pseudoalteromonas antartica, is thought to induce nucleation of ice and prevent the growth of large ice crystals thus allowing the microorganism to survive in the Antarctic environment. Antarticine-NF3 has been shown to be useful for the treatment and re-epithelialisation of wounds in the pharmaceutical and veterinary fields, as well as for cosmetic regeneration treatments (US 2004/0242466).

[0105] In other embodiments the desirable compounds include secondary metabolites (such as enzymes, immunomodulatory agents, chemotherapy agents, cytotoxins, antibiotics, antifungal and antiviral agents), typically produced during the stationary growth phase (idiophase) of a microorganism culture. Secondary metabolites are non-growth related and seem to play no obvious role in cell maintenance. When the objective is to produce secondary metabolites, the aim of fermentation will be to maximize the biomass in a short growth phase, while optimizing the conditions for high, sustained idiophase production.

[0106] Desired compounds include a number of heterocyclic compounds such as NPI-0052 produced during the fermentation of Salinospora tropica, a marine Gram- positive actinomycete (WO 05/002572). These compounds are useful as proteasome inhibitors in the treatment of inflammation, cancer, and infectious diseases

[0107] Following incubation the medium is harvested for processing and isolation of the desired microorganism or compounds. Harvesting, processing and isolation can be carried out using known art protocols. [0108] Harvesting includes the step of inactivating the microorganisms, either before or after removal of the medium from the fermenter vessel.

[0109] In one embodiment the saline fermentation medium is inactivated in situ in the fermenter vessel by gentle heating at about 70⁰C for up to about 2 hours. Other options for inactivating include addition of chemical biocides or solvents, physical removal (e.g. by filtration or centrifugation), or a combination of these techniques.

[0110] Corrosion can be further minimized by transferring the saline fermentation medium to a separate, highly corrosion resistant inactivation process vessel such as a heat exchanger.

[0111] The desired compounds can then be isolated from the disrupted cells using standard techniques known in the art.

[0112] When the fermenter vessel is to be reused it is rinsed to lower the salt concentration and is preferably sterilized before being visually inspected for evidence of corrosion. Any corrosion present is removed using mechanical abrasion, using abrasive paper or an abrasive pad.

[0113] In preferred embodiments the surface of the fermenter vessel is restored to a 320 grit finish using 320 grit wet silicon carbide paper.

[0114] The fermenter vessel is then repassivated using a nitric acid solution to enhance the formation of a passive protective film, ready for subsequent fermentation processes.

[0115] The invention will now be described in greater detail by reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Example 1 Exposure of Grade 304 and 316 stainless steel coupons to simulated fermentation conditions

[0116] Pitting or crevice corrosion of stainless steels generally requires the presence of a halide species, usually chloride, and will occur when the open-circuit potential (OCP) exceeds the pitting potential (E_pu) or crevice corrosion potential (E_crev). The OCP for stainless steels is determined primarily by the oxidizing power of the solution, while Epu or E_crev depend primarily on the temperature and the concentrations of the various anions present in the solution. Figures 1 and 2 show laboratory data for the pitting potentials of grade 302, 304 and 316 stainless steels as functions of temperature and chloride concentration. Natural seawater produces biofilms or bacteria or algae that generate maximum OCP values of up to 300 mV, well above the pitting potentials of these stainless steels.

[0117] From the literature, grade 304 has adequate pitting resistance in water containing less than 200 ppm chloride, but its performance is marginal when chloride concentration is between 200 ppm and 1,000 ppm chloride. 316 grades have adequate pitting resistance up to about 1,000 ppm chloride. Accordingly, 304 and 316 would not normally be considered suitable for saline fermentations.

[0118] The Pitting Resistance Equivalent (PRE) number is considered to be a good indication of the pitting resistance of stainless steels. The PRE can be calculated as: PRE = %Cr + 3.3 x %Mo + 16 x %N. Grades high in chromium, and particularly molybdenum and nitrogen, are thus more resistant to pitting corrosion.

[0119] It is generally accepted that stainless steel having a PRE number greater than 40 is necessary for stainless steel to have good resistance to pitting corrosion in seawater, able to withstand long periods of stagnation at temperatures not exceeding 40⁰C. A PRE number greater than 45 is considered necessary to have good resistance to crevice corrosion in natural seawater. Type 304 grade stainless has a PRE of 19, whereas the PRE of Type 316 is 25, both well below that thought necessary to provide good corrosion resistance to seawater.

[0120] To determine the degree of likely corrosion of stainless steel, two types of tests were carried out:

• exposure of prepared coupons in artificial seawater (Instant Ocean®); and Instant Ocean® plus growth media;

• electrochemical measurements of pitting properties of the primary alloy of interest, type 316L at two temperatures of interest (28°C and 50⁰C).

[0121] The aims of the coupon exposures were to mimic an actual fermentation exposure cycle, to determine the pits that would be initiated on 320 grit prepared surfaces during:

• extended exposures conducted at 28°C,

• short term exposures at high temperatures required for downstream processing, 55⁰C and 121⁰C.

[0122] The main aims of the electrochemical testing were to determine whether the presence of bacterial growth media would increase or decrease the pitting potential and the extent to which the increase in temperature from 28°C to 50⁰C would lower the pitting potential (and therefore increase the risk of pitting corrosion in service) for type 316L in saline solutions.

Materials Tested

• Type 304 stainless steel.

• Type 304 welded stainless steel.

• Type 316L stainless steel.

• Type 316L welded stainless steel.

[0123] The surface of the coupons was polished using dry, rotating 320 grit paper followed by water wash, ethanol wash and swab, ethanol rinse and warm air dry. The coupons were desiccated overnight and weighed twice to 0.00001 g prior to exposure.

Process Variations Tested

• Separate sterilization of fluids and materials prior to mixing for exposure.

• Exposure at 28⁰C for 7 days under stirred conditions to individual solutions of: - Instant Ocean® (30 g/L).

Instant Ocean® + growth media, prepared according to the following concentrations (at tolerances of ± 2.5%):

SHY 10-4-4 (Production) Cl^' component

USB Starch 1O g 0

USB Yeast Extract 4 g 0.1 g

Kerry Hy Soy 4 g 0.11 g

Schlau CaCO₃ I g 0

Fe₂(SO4)₃ 40 mg 0

KBr lOO mg 0

Instant Ocean® 30 g 16.5 g

Purified Water 1 L 0

Clerol FBA 5059 0.1 g/L 0

TOTAL 16.71 g/L CI^"

Instant Ocean® + growth media + microorganisms. Incubation + sterilization at 55°C for 15 minutes. • Incubation + sterilization at 55°C + sterilization at 121⁰C for 45 minutes.

[0124] The exposure trials were conducted in non-baffled 2 L Erlenmeyer flasks with the coupons positioned by Teflon tube having a tight fit in the 4mm diameter hole drilled in one end of each coupon. 900 mL of solution was used, and the flasks stoppered with cotton wool. The flasks were held at 28°C in a water bath and shaken with a 50 mm throw at 95 rpm to ensure aeration.

Results Summary

[0125] The coupon exposure trials show minimal changes in mass between the tested stainless steels when exposed in the above solutions. Grade 304 coupons were found to have an average mass change of -0.1 mg to 0.1 mg, whilst the mass change of the grade 316L coupons was from -0.49 mg to +0.35 mg. No one material or process step resulted in consistent mass loss or mass gain.

[0126] Minimal evidence of surface corrosion was found on either the grade 304 or grade 316 L coupons.

[01271 The electrochemical testing work in laboratory simulated solutions with "bacterial growth media" added to Instant Ocean® solution confirmed Type 316L stainless steel was a borderline material in these environments, particularly in stagnant or low flow situations. Addition of growth media gave inconsistent results, being detrimental at 28°C and beneficial at 50⁰C.

[0128] As expected from the literature, increasing the temperature of the incubation from 28°C to 50⁰C shifted the measured pitting distributions to lower potential values, as shown in Figure 3. Increasing the temperature thus increases the risk of pitting corrosion.

[0129] At 28⁰C, about 50% of the measured pitting potentials were about 150 mV or above. Comparing these results to an estimated maximum OCP of 150 mV (vs SCE) for stainless steel in artificial seawater is consistent with the hypothesis of 316L being a borderline material at this temperature.

[0130] At 50⁰C however, all the measured pitting potentials. were below 100 mV, suggesting a high risk of pitting corrosion for 316L in artificial seawater at this temperature, particularly in low flow or stagnant conditions. Example 2

Full fermentation run and post inspection assessment

[01311 The aim of these experiments was to complete two full fermentation process runs in conjunction with an inspection and monitoring program for corrosion before, during and after each run.

[0132] The fermentation process was conducted using the\ NPI-0052 producing organism Salinospora tropica, as described in WO 05/002572. [0133] Three different vessels were used:

• Vessel Fer 001 , a Type 316L welded 300 L fermentation tank.

• Vessel Fer 003, a Type 316L welded 1500 L fermentation tank.

• Vessel KiI 001, a Type 304 welded 300 L kill tank.

[0134] The first full process run was completed using the following vessel/process combinations:

• Fer 001, used for seed fermentation for two days with full mix at 28°C with the stirrer speed set at 70 rpm, prior to Fer 003.

• Transfer pipe used to move solution to Fer 003.

• Fer 003 inoculation followed by five days at 28°C with the stirrer speed set at 60 rpm. 450 L Instant Ocean® solution (60 g/L) was added to the tank at the beginning of the fermentation to give 900 L of fermentation broth of composition 30 g/L Instant Ocean®. Therefore the composition of the saline fermentation media was prepared according to the following concentrations, at tolerances of ± 1%:

SHY 10-4-4 (Production) CT component

USB Starch 1O g 0

USB Yeast Extract 4 g 0.1 g

Kerry Hy Soy 4 g 0.11 g

Schlau CaCO₃ I g 0

Fe₂(SO4)₃ 40 mg 0

KBr 100 mg 0

Instant Ocean® 30 g 16.5 g

Purified Water 1 L 0

Clerol FBA 5059 0.1 g/L 0

TOTAL 16.71 g/L CT • Following fermentation the media was harvested and the vessel rinsed with purified water before being sterilized at 121°C.

• Transfer pipe used to move solution to KiI 001.

• KiI 001, used at 121⁰C for one hour followed by slow cool to 80⁰C, followed by overnight cool to 40 to 60⁰C, followed by dilution with flush waters.

Vessel Exposure Risks

[0135] It was previously hypothesized that the greatest risks in the proposed processing would be:

• If the solutions were allowed to sit without stirring for an extended period.

• If the process times at higher temperatures were extended.

• If the process allowed deposit formation on the vessel walls giving conditions that promote crevice corrosion.

[0136] The inspection and monitoring program included:

• Condition of the exposed vessel internals before and after exposure:

Appearance of polish.

Apparent cleanliness.

CIP records.

Deposit formation and distribution.

Appearance of metal under any deposits.

• Monitoring of actual exposure conditions including:

Exposure temperatures/pressures as a function of time.

Stirring condition and efficacy.

Concentrations of key solution components.

Dissolved species marker concentrations, i.e. chromium, nickel.

Vessel Surface Inspection Results

[0137] The inspections conducted are summarized as follows: Fer 001, two days at 28°C with full mix.

• The underside of the flat lid was wet when the vessel was opened for inspection. There were mechanical damage marks in a few areas; none appeared to be actively corroding. There were minor superficial "rust" stain areas from standby corrosion (i.e. these stains likely formed after the run was completed).

• Vessel had four internal vanes running vertically, an external water jacket and ports on the lower floor area for air sparging. A single stirrer was used during the process. There was an area of white stain-like deposit about 300 mm below the level of the top of the water jacket, possibly at a water line. There was no evidence of rust staining on the vessel walls or lower dome.

Fer 003, five days at 28°C with full mix after inoculation. ^

• The domed lid had drainage marks at some ports suggesting deposits from solution. There was a single pit next to one of the ports; this was measured to be 0.001 inch deep using a pit depth micrometer (0.025 mm). The pit was rust stained.

• The vessel had four vanes running vertically, a number of side ports top and bottom, air sparge from the bottom, a single stirrer. The surface finish was obtained using varying grades of abrasive pads before the run and appeared to be of sufficient quality for the vessel; there were retained mechanical surface defects but no apparent active corrosion. A few "rust" stain areas associated with grey/white scale deposits were noted on the side wall near the top of one of the paddles, these were superficial and easily rubbed off. There were some other similar "rust" stain areas away from grey/white scale deposits, again on the side walls.

• There was evidence of a warm spot on the jacket suggesting steam valve leakage.

Discussion

[0138] The results of the previously completed coupon exposures and laboratory tests described in Example 1 indicated a lesser risk of localized corrosion with the Instant Ocean® + media + microorganisms than would have been predicted with natural sea water. The Type 304 and 316L coupons gave minor indication of possible added corrosion at sites of mechanical defects but the evidence was low.

[0139] Inspections conducted on Fer 001 and Fer 003 vessels indicated minimal adverse effects from the first full fermentation process run. [0140] The one measurable pit observed in the lid of Fer 003 had a depth of 0.025 mm and was most likely present prior to the run. There is a high likelihood that the observed rust stain at this pit site was indicative of some active corrosion but this may have developed under standby conditions after the run had been completed rather than on-line. Superficial rust stains in other parts of the Fer 003 vessel were also most likely the result of standby corrosion at the end of the run.

[0141] Deposit staining was observed in both Fer 001 and Fer 003 and it is possible that this staining occurred at the end of the run when the solutions were being transferred.

[0142] KiI 001 tank could not be internally inspected as the vessel was in use. Evidence of flange leakage, most likely at an earlier date was seen as deposits in the flange area.

Conclusions

[0143] The Fer 001 and Fer 003 vessels used showed minimal evidence of localized corrosion as a result of exposure to the process solutions in the first run. Deposit staining was observed in both these vessels but was believed due to evaporative concentration at the end of the run when the solutions were transferred. Superficial rust staining was seen in Fer 003 and was likely due to standby corrosion at the end of the run.

[0144] The general view held in the industry and literature is that stainless steel vessels are unsuitable for saline fermentation protocols. This view was confirmed by the coupon trials of Example 1 where grade 316 was shown to be borderline at 28°C and at high risk of corrosion at 50 ⁰C.

[0145] The present inventors have, however, surprisingly found that stainless steel fermenters can be used for saline fermentation, with corrosion being minimized during fermentation by minimizing the time the chloride salt solution is in contact with the stainless steel at elevated temperatures and ensuring the medium is kept in motion at all times to prevent formation of biofilms. Where the vessels are to be reused they should be inspected at the end of each run and any corrosion present removed and the vessel repassivated to ensure an optimal surface finish to reduce future corrosion and pitting. INDUSTRIAL APPLICATION

[0146] The elevated temperatures involved in sterilization and fermentation of high chloride media results in the rapid corrosion of stainless steel vessels to such an extent that stainless steel is generally viewed as unsuitable for saline fermentation protocols.

[0147] The present invention is directed to methods of minimizing the corrosion caused by saline solutions to the stainless steel fermenter vessels. The inventors have shown that stainless steel fermenters can be used for saline fermentation, with corrosion being minimized during fermentation by minimizing the time the chloride salt solution is in contact with the stainless steel at elevated temperatures and ensuring the medium is kept in motion at all times to prevent formation of biofilms.

[0148] Those persons skilled in the art will understand that the above description is provided by way of illustration only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims

WHAT WE CLAIM IS:

1. A saline fermentation method comprising:

(a) charging a fermenter vessel with a fermentation medium having less than about 300 ppm chloride ions;

(b) sterilizing the medium in the vessel;

(c) adding a sterile chloride salt solution to the medium to produce a saline fermentation medium;

(d) inoculating the saline fermentation medium with a microorganism and culturing the saline fermentation medium under conditions suitable for the growth of the microorganism; and

(e) harvesting the medium.

2. A method of minimizing corrosion of a stainless steel vessel during saline fermentation of a microorganism, comprising:

(a) charging a fermenter vessel with a fermentation medium having less than about 300 ppm chloride ions;

(b) sterilizing the medium in the vessel;

(c) adding a sterile chloride salt solution to the medium to produce a saline fermentation medium;

(d) inoculating the saline fermentation medium with a microorganism and culturing the saline fermentation medium under conditions suitable for the growth of the microorganism; and

(e) harvesting the medium.

3. The method of claim 1 or 2, wherein the medium is heat sterilized.

4. The method of claim 3, wherein the medium is heat sterilized at a temperature between about 75°C to about 140⁰C.

5. The method of claim 3, wherein the medium is heat sterilized at a temperature of about 121⁰C .

6. The method of claim 3, wherein the medium is heat sterilized for a period of from about 15 minutes to about 45 minutes.

7. The method of claim 1 or 2, wherein the fermenter vessel is constructed of stainless steel.

8. The method of claim 7, wherein the stainless steel is a 300 series grade stainless steel.

9. The method of claim 7, wherein the stainless steel has a pitting resistance equivalent number less than about 45.

10. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 6,500 ppm and about 90,000 ppm.

11. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 1 1 ,000 ppm and about 60,000 ppm.

12. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 12,000 ppm and about 5O₅OOO ppm.

13. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 13,000 ppm to about 40,000 ppm.

14. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 14,000 ppm and about 30,000 ppm.

15. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration between about 15,000 ppm and about 20,000 ppm.

16. The method of claim 1 or 2, wherein the saline fermentation medium has a chloride ion concentration of about 18,000 ppm.

17. The method of claim 1 or 2, wherein the chloride salt solution comprises one or more chloride salts selected from the group consisting of calcium chloride (CaCl₂), cobalt chloride (CoCl₂), cesium chloride (CsCl), potassium chloride (KCl), lithium chloride (LiCl), magnesium chloride (MgCl₂), manganese chloride (MnCl₂), sodium chloride (NaCl), ammonium chloride (NH₄Cl), nickel chloride (NiCl₂), rubidium chloride (RbCl), zinc chloride (ZnCl₂), sodium perchlorate (NaClO₄), and ferric chloride (FeCb).

18. The method of claim 1 or 2, wherein the chloride salt solution comprises sodium chloride (NaCl).

19. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 1 1 g/L and about 150 g/L.

20. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 18 g/L and about 100 g/L.

21. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 20 g/L and about 83 g/L.

22. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 21.5 g/L and about 67 g/L.

23. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 23 g/L and about 50 g/L.

24. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is between about 24 g/L and about 33 g/L.

25. The method of claim 18, wherein the sodium chloride concentration of the saline fermentation medium is about 30 g/L.

26. The method of claim 1 or 2, wherein the microorganism comprises at least one microorganism selected from the group comprising bacteria, protozoa, algae, cyanobacteria, yeast and fungus.

27. The method of claim 26, wherein the microorganism comprises at least one microorganism selected from the group comprising is Actinopolyspora, Haloferax, Halobacterium, Halobacillus, Marinilactibacillus, Spirochaeta, Vibrio, Pseudomonas, Haloarcula, Rhodothermus, Halomonas, Halococcus, Salinospora, Anaerophaga, Haloanaeroba, Debaryomyces, Scytalidium, Hyphomycetes, Zygomycetes, Ascomycetes, Trimmatostroma, and Hortaea.

28. The method of claim 26, wherein the microorganism comprises at least one of the following two microorganisms: Salinospora tropica or Pseudoalteromonas antarctica.

29. The method of claim 1 or 2, wherein the growth is conducted at a temperature between about 10⁰C and about 70 ⁰C.

30. The method of claim 1 or 2, wherein the growth is conducted at a temperature between about 20⁰C and about 65°C.

31. The method of claim 1 or 2, wherein the growth is conducted at a temperature between about 25°C and about 50⁰C.

32. The method of claim 1 or 2, wherein the growth is conducted at a temperature between about 26°C and 40⁰C.

33. The method of claim 1 or 2, wherein the growth is conducted at a temperature of about 28°C.

34. The method of claim 1 or 2, wherein the pH of the saline fermentation medium is between about pH 1 and about pH 11.

35. The method of claim 1 or 2, wherein the pressure under which saline fermentation occurs is between about 1 and about 100 bar.

36. The method of claim 1 or 2, wherein the saline fermentation medium is in motion during fermentation.

37. The method of claim 36, wherein the motion is achieved through mechanical mixing.

38. The method of claim 36, wherein the motion is achieved through gas- sparging.

39. The method as claimed in any one of claims 1 to 38, wherein the fermentation is aerobic.

40. The method of claim 1 or 2, wherein the fermentation is anaerobic.

41. The method of claim 1 or 2, further comprising the step on inactivating the saline fermentation medium.

42. The method of claim 41, wherein inactivating is achieved by heating.

43. The method of claim 41, wherein the inactivating in achieved by addition of a chemical biocide.

44. The method of claim 41, wherein the harvesting and inactivating steps are conducted, at least in part, in a separate process vessel.

45. The method of claim 44, wherein the separate process vessel is a heat exchanger.

46. The method of claim 1 or 2, comprising the further steps of:

(i) inspecting the vessel after use and removal of any corrosion present; and

(ii) repassivating the vessel.

47. The method of claim 46, wherein the corrosion comprises at least one of pitting corrosion, crevice corrosion and stress corrosion cracking.

48. The method of claim 47, wherein any corrosion present after fermentation is removed by mechanical abrasion.

49. The method of 46, wherein the vessel is repassivated through the use of nitric-acid.