(12) United States Patent (10) Patent No.: US 9,580,738 B2 Han Et Al

USOO9580738B2

(12) United States Patent (10) Patent No.: US 9,580,738 B2 Han et al. (45) Date of Patent: *Feb. 28, 2017

(54) METHOD FOR PRODUCING OTHER PUBLICATIONS EXTRACELLULAR PROTEINS FROM GENUS TEPIDEMONAS Franca et al. International J of Systematic and Evolutionary Micro biology, 2006, 56:907-912.* (71) Applicant: INDUSTRIAL TECHNOLOGY “Microorganisms; 133. Carbon Monoxide Oxidizer Medium.” RESEARCH INSTITUTE, Hsinchu DSMZ GmbH, 2009, 1 page. (TW) “Microorganisms; 27. Rhodospirillaceae Medium (modified).” DSMZ GmbH, 2007, 1 page. (72) Inventors: Yin-Lung Han, Tainan (TW); Jo-Shu Taiwanese Office Action and Search Report, dated Jan. 12, 2016, for Chang, Taichung (TW); Yung-Chong Taiwanese Application No. 103145419. Wan-Ju Yu et al., “Optimal environmental condition and medium Lou, Tainan (TW); Chieh-Lun Cheng, composition for protein production of T. fonticaldi AT-A2.” Con Taoyuan (TW); Chih-Hsi Liu, Hsinchu ference on Biotechnology and Bioengineering of BEST, Jun. 28. (TW) 2013, 4 pages. Albuquerque et al., “Tepidimonas thermarum sp. nov... a new (73) Assignee: INDUSTRIAL TECHNOLOGY slightly thermophilic betaproteobacterium isolated from the RESEARCH INSTITUTE, Hsinchu Elisenguelle in Aachen and emended description of the genus (TW) Tepidimonas', Systematic and Applied Microbiology 29, 2006, pp. 450-456. (*) Notice: Subject to any disclaimer, the term of this Chen et al., “Tepidimonas fonticaldi sp. nov., a slightly thermophilic patent is extended or adjusted under 35 betaproteobacterium isolated from a hot spring. International Jour U.S.C. 154(b) by 0 days. nal of Systematic and Evolutionary Microbiology, 2013, 63, pp. 1810-1816. This patent is Subject to a terminal dis Chen et al., “Tepidimonas taiwanensis sp. nov., a novel alkaline claimer. protease-producing bacterium isolated from a hot spring'. Extremophiles, 2006, pp. 35-40. (21) Appl. No.: 14/838,612 Moreira et al., “Tepidimonas ignava gen, nov... sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the (22) Filed: Aug. 28, 2015 B-Proteobacteria” International Journal of Systematic and Evolu tionary Microbiology, 2000, 50, pp. 735-742. (65) Prior Publication Data US 2016/0186226 A1 Jun. 30, 2016 * cited by examiner

(30) Foreign Application Priority Data Primary Examiner — Bin Shen Dec. 25, 2014 (TW) ...... 103145419 A (74) Attorney, Agent, or Firm — Birch, Stewart, Kolasch & Birch, LLP (51) Int. Cl. CI2P 2L/00 (2006.01) (52) U.S. Cl. (57) ABSTRACT CPC ...... CI2P21/00 (2013.01) The disclosure provides a method for producing extracellu (58) Field of Classification Search lar proteins from genus Tepidimonas, including: performing CPC ...... C12P 21 FOO a fermentation culturing to a bacteria of genus Tepidimonas See application file for complete search history. with a culturing medium to form a fermented liquid, wherein the composition of the culturing medium includes a carbon (56) References Cited Source which is an organic acid, selected from a group consisting of acetate, lactate and butyrate; a nitrogen Source U.S. PATENT DOCUMENTS selected from a group consisting of (NH4)2SO. NHNO. 8,129,155 B2 * 3/2012 Trawick ...... C12N 9,0006 NHCl and urea; phosphate; carbonate; MgCl, yeast 435/146 extract; and trace elements, and wherein the gas feeding rate 8,728,777 B2 5, 2014 Biton et al. for the fermentation culturing is about 0-0.1 VVm; and after 2004/01754O7 A1 9, 2004 McDaniel the fermentation culturing is completed, collecting the fer 2012fOO58533 A1 3/2012 Biton et al. mented liquid, wherein the fermented liquid contains extra FOREIGN PATENT DOCUMENTS cellular protein secreted from the bacteria of genus Tepidi FiOS CN 102628O25 A 8, 2012 TW 201328597 A T 2013 TW 201348.442 A 12/2013 38 Claims, 6 Drawing Sheets U.S. Patent Feb. 28, 2017 Sheet 1 of 6 US 9,580,738 B2

U.S. Patent Feb. 28, 2017 Sheet 2 of 6 US 9,580,738 B2

S U.S. Patent Feb. 28, 2017 Sheet 3 of 6 US 9,580,738 B2

1. 2 D Protein production (mg/L) 1. O ... Protein production rate (mg/L/day) 8

Temperature (C) FIG. 2

O Protein production (mg/L) 3 O ... Protein production rate (mg/L/day) I 1 1 2 0

O 0.0125 0.025 Air feeding rate (vvm) FIG. 3 U.S. Patent Feb. 28, 2017 Sheet 4 of 6 US 9,580,738 B2

O Protein production (mg/L) ... Protein production rate (mg/L/day) O Yield (mg protein/g acetate)

16 14 30 14 12 12 10 10 2 O

Directly Together Separate air air air feeding feeding feeding from with liquid liquid circulation circulation Different air feeding type

FIG. 4 U.S. Patent Feb. 28, 2017 Sheet S of 6 US 9,580,738 B2 25 as O Protein production (mg/L) s & Protein production rate (mg/L/day) 20 g 15 s 8 5 O5. 10 5r 9 w 5 s 9 . Al- S2

7.5 8.0 8.5 0 Final pH controlled FIG. 5

O Protein production (mg/L) ... Protein production rate (mg/L/day) 23 OO

O 1.5 3.0 6.0 Liquid recycle rate (L/h) FIG. 6 U.S. Patent Feb. 28, 2017 Sheet 6 of 6 US 9,580,738 B2

O Protein production (mg/L) x Protein production rate (mg/L/day) o Yield (mg protein/g acetate) 14 10 3. 12 5 40 8 10 5 6 : 8 30 9 g .9 6,O 20 4 rg E S rt 45 2 E. is E 10 .S > A. & 3. & 32 O O is: O S.OL 100 100 100 (OD=0.03) (2.5%) (5%) (10%) Different inoculation concmetration FIG. 7 US 9,580,738 B2 1. 2 METHOD FOR PRODUCING FIG. 1A shows an air feeding type directly feeding the air EXTRACELLULAR PROTEINS FROM into the bottom of the culture medium according to an GENUS TEPIDEMONAS embodiment of the disclosure; FIG. 1B shows an air feeding type according to another CROSS REFERENCE TO RELATED embodiment of the disclosure, circulating the culture APPLICATION medium by introducing the medium out from the fermenter and sending it back into the fermenter and, during the The present application is based on, and claims priority circulation, the air is mixed into the culture medium before from, Taiwan Application Serial Number 103145419, filed it is sent back; on Dec. 25, 2014, the disclosure of which is hereby incor 10 FIG. 1C shows an air feeding type according to another porated by reference herein in its entirety. embodiment of the disclosure, circulating the culture medium by introducing the medium out from the fermenter TECHNICAL FIELD and sending it back into the fermenter and feeding the air 15 into the top of the fermenter; The technical field relates to a method for producing FIG. 2 shows the protein yields and production rates of extracellular proteins from genus Tepidimonas. culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at BACKGROUND different culturing temperatures according to one embodi ment of the disclosure; The inventors of the present disclosure have sampled the FIG. 3 shows the protein yields and production rates of water samples of An-tong hot spring in Hualien County, culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at Taiwan. Then, a novel bacterium of Tepidimonas named different air feeding rate according to one embodiment of the Tepidimonas fonticaldi sp. nov. in the water samples was disclosure; isolated and purified. Extracellular proteins secreted by FIG. 4 show the protein yields, production rates and per Tepidimonas fonticaldi sp. nov. have excellent effects for 25 gram of acetate of protein yields of culturing Tepidimonas binding metal ions, and are not influenced by environmental fonticaldi sp. nov. KCTC 12528BP at different air feeding conditions, such as high temperature, high pressure, or pH types according to one embodiment of the disclosure; value. Therefore, the extracellular proteins secreted by Tepi FIG. 5 shows the protein yields and production rates of dimonas fonticaldi sp. nov. can prevent metal salt scaling, culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at especially calcium carbonate, from being formed in boiler 30 different final pH of the fermenting according to one equipment, underground pipelines, geothermal wells, indus embodiment of the disclosure; trial wastewater or hard water, to maintain the proper FIG. 6 shows culturing Tepidimonas fonticaldi sp. nov. functioning of the machines and reduce operating time and KCTC 12528BP circulating the culture medium by intro COStS. ducing the medium out from the fermenter and sending it Bacteria of genus Tepidimonas are bacteria which are 35 back into the fermenter and, during the circulation, the air is gram-negative, strictly aerobic, oxidase- and catalase-posi mixed into the culture medium before it is sent back at tive, rod-shaped and slightly thermophilic bacteria. different circulation rates of the protein yields and produc However, the growth and self-protein metabolizing rate of tion rates of culturing Tepidimonas fonticaldi sp. nov. KCTC bacteria of genus Tepidimonas are slow, and this makes it 12528BP according to one embodiment of the disclosure, hard to obtain the protein. Therefore, a novel method of 40 and producing extracellular proteins from genus Tepidimonas is FIG. 7 shows the protein yields and production rates of needed. culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at different inoculation concentrations according to one SUMMARY embodiment of the disclosure. 45 The disclosure provides a method of producing extracel DETAILED DESCRIPTION lular proteins from genus Tepidimonas, including ferment ing a Tepidimonas with a culture medium in an air feeding In the following detailed description, for purposes of type to obtain a suspension; wherein the culture medium has explanation, numerous specific details are set forth in order a carbon Source which is an organic acid selected from a 50 to provide a thorough understanding of the disclosed group consisting of acetate, lactate and butyrate; a nitrogen embodiments. It will be apparent, however, that one or more Source selected from a group consisting of ammonium embodiments may be practiced without these specific sulfate ((NH4)2SO), ammonium nitrate (NHANO), ammo details. In other instances, well-known structures and nium chloride (NHCl) and urea; a phosphate; a carbonate; devices are shown schematically in order to simplify the magnesium chloride (MgCl); a yeast extract; and trace 55 drawings. elements; and collecting the Suspension, wherein the Sus In one embodiment of the present disclosure, a method of pension includes the extracellular proteins from genus Tepi producing extracellular proteins from genus Tepidimonas is dimonas; and wherein the air feeding condition is about provided. The extracellular proteins can effectively be 0-0.1 VVm air feeding rate. obtained from the genus Tepidimonas fonticaldi by using the A detailed description is given in the following embodi 60 methods of the present embodiments. ments with reference to the accompanying drawings. A method of producing extracellular proteins from genus Tepidimonas may include the following steps, but it is not BRIEF DESCRIPTION OF DRAWINGS limited thereto. First, fermenting a Tepidimonas with a culture medium in The disclosure can be more fully understood by reading 65 an air feeding type to obtain a suspension. In one embodi the Subsequent detailed description and examples with ref ment, the bacterium of genus Tepidimonas is cultured for erences made to the accompanying drawings, wherein: about 48-270 hrs. US 9,580,738 B2 3 4 Examples of the bacterium of genus Tepidimonas men the content of the phosphate may be about 0.1 g/L-30 g/L, tioned above may comprise, but are not limited to Tepidi the content of the carbonate may be about 0.1 g/L-20 g/L, monas fonticaldi, Tepidimonas ignava, Tepidimonas the content of the MgCl2 may be about 0.01 g/L-10 g/L, the aquatic, Tepidimonas taiwanesis, etc. content of the yeast extract may be about 0.1 g/L-20 g/L, and In one embodiment, a bacterium of genus Tepidimonas of 5 the content of the trace elements may be about 0.01 g/L-1 the present disclosure is Tepidimonas fonticaldi. The Tepi g/L, but it is not limited thereto. dimonas fonticaldi is Tepidimonas fonticaldi sp. nov., which In the culturing medium of one embodiment, the carbon was deposited at the Laboratorium voor. Microbiologie nitrogen weight ratio of the carbon Source to the nitrogen Gent Belgium (LMG) on Nov. 21, 2011, and the deposit source may be about 1-60, and in another embodiment, the number is LMG26746. 10 carbon-nitrogen weight ratio of the carbon Source to the In another one embodiment, a bacterium of genus Tepi nitrogen source may be about 11. dimonas of the present disclosure is Tepidimonas fonticaldi. In one embodiment, the fermenting step is conducted in a The Tepidimonas fonticaldi is Tepidimonas fonticaldi sp. fermenter, but it is not limited thereto. In an exemplary nov., which was deposited at the Korean Collection for Type embodiment, liquid capacity of the fermenter is 5 L. In Cultures (KCTC) on Dec. 4, 2013, and the deposit number 15 another embodiment, liquid capacity of the fermenter is 100 is KCTC 12528BP. The Tepidimonas fonticaldi sp. nov. L. In another embodiment, liquid capacity of the fermenter KCTC 12528BP is capable of producing extracellular pro may be is ton-level. teins. The fermenting step comprises a stirring step, in which The culturing medium for culturing a bacterium of genus the stirring rate may be about 50-500 rpm, and in another Tepidimonas of the present disclosure may comprise, but is 20 embodiment, the stirring rate may be about 200 rpm, but it not limited to, a carbon source, a nitrogen source, phosphate, is not limited thereto. magnesium chloride (MgCl), yeast extract and trace ele In the fermenting step the bacterium of genus Tepidimo mentS. nas is cultured at about 40-80° C. In one embodiment, the The culturing medium of genus Tepidimonas of the pres bacterium of genus Tepidimonas is cultured at about 50° C. ent disclosure is not limited to, but may comprise, a carbon 25 The fermenting step conducted in a fermenter may com Source, a nitrogen source, a phosphate, a carbonate, mag prise feeding air into the culture medium, or it may not be nesium chloride (MgCl), a yeast extract and trace elements. necessary. When air feeding, the air feeding rate for the In some embodiments, the above-mentioned carbon fermenting is about 0-0.1 VVm, but it is not limited thereto. Source for the culturing medium is an organic acid, and Furthermore, when the air feeding rate is 0 VVm, it means the examples of the above-mentioned organic acid may com- 30 fermenting step is performed without air feeding. In one prise, but are not limited to, acetate, lactate, butyrate, etc. In embodiment, the gas used for the air feeding may comprise, one embodiment, the above-mentioned carbon source may but is not limited to, air. be acetate. In one embodiment, the fermenting is conducted in a Moreover, in some embodiments, the above-mentioned fermenter, and the air feeding condition may comprise three nitrogen source for the culturing medium may comprise 35 feeding types, shown in FIGS. 1A, 1B and 1C, but it is not ammonium Sulfate ((NH4)2SO4), ammonium nitrate limited thereto. (NHNO), ammonium chloride (NHCl), urea, etc., but it is In the first air feeding type, air is fed directly into the not limited thereto. In one embodiment, the above bottom of the culture medium. As shown in FIG. 1A, the air mentioned nitrogen source may be ammonium Sulfate is fed into the bottom of the culture medium 103 via piping ((NH)SO). 40 101 extending from the top of the fermenter 100. In some embodiments, the suitable phosphate for the The second air feeding type involves circulating the culturing medium may comprise, but is not limited to, culture medium by introducing the medium out from the disodium hydrogen phosphate (NaHPO), potassium dihy fermenter and sending it back into the fermenter and, during drogen phosphate (KH2PO4), Sodium dihydrogen phosphate the circulation, the air is mixed into the culture medium (NaH2PO), dipotassium phosphate (KHPO), etc. or any 45 before it is sent back. FIG. 1B shows how the culturing combination thereof. medium 103 is introduced from the fermenter 100 via an In some embodiments, the carbonate for the culturing internal piping 105 and sent back to the fermenter 100 via an medium may comprise, but is not limited to, sodium bicar external piping 107 to form an outer liquid circulation 111, bonate (NaHCO). and air is mixed into the culturing medium 103 before it is Furthermore, the trace elements for the culturing medium 50 sent back via piping 113. may comprise, for example, Zinc (Zn), manganese (Mn), The third air feeding type is circulating the culture boron (B), cobalt (Co), copper (Cu), nickel (Ni), molybde medium by introducing the medium out from the fermenter num (Mo), etc., but is not limited thereto. In one embodi and sending it back into the fermenter and feeding the air ment, the trace elements in the culturing medium of the into the top of the fermenter. FIG. 1C shows how the present disclosure may comprise Zinc, manganese, boron, 55 culturing medium is introduced out from the fermenter via cobalt, copper, nickel and molybdenum. In this embodiment, the internal piping 105 and culturing medium via the exter the respective element may be in the forms shown in the nal piping 107 to form an outer liquid circulation 111 that is following, but is not limited thereto: zinc may be in the form sent back to the fermenter via another external piping 109, of Zinc sulfate (ZnSO), manganese may be in the form of and the air is fed into the top of the culture medium 103 via manganese chloride (MnCl), boron may be in the form of 60 piping 115. boric acid (HBO), cobalt may be in the form of cobalt In one embodiment, the air feeding type involves directly chloride (CoCl2), copper may be in the form of copper feeding a gas into the bottom of the culture medium, and the chloride (CuCl), Ni may be in the form of nickel chloride gas is air. In one example, the air feeding rate for the (NiCl), and Mo is sodium molybdate (Na2MoC). fermenting is about 0.001-0.1 VVm, or it may be about 0.025 In the culturing medium of one embodiment, the content 65 VV. of the carbon source may be about 0.1 g/L-15 g/L, the In one embodiment, the air feeding type involves circu content of the nitrogen source may be about 0.1 g/L-15 g/L. lating the culture medium by introducing the medium out US 9,580,738 B2 5 6 from the fermenter and sending it back into the fermenter culturing temperatures for a 5 L fermenter. The testing and, during the circulation, a gas is mixed into the culture temperatures were 45° C., 50° C., and 55° C. medium before it is sent back, in one embodiment in which The Tepidimonas KCTC 12528BP was cultured by a the gas is air, and the air feeding rate for the fermenting is medium shown in the following Table 1. In the culturing about 0.001-0.1 VVm, or it may be about 0.025 VVm, and the medium, the concentration of acetate was 3.11 g/L, ammo medium circulation ratio is about 0.1-12 L/hr, or it may be nium Sulfate was 0.53 g/L, and the carbon-nitrogen weight about 3 L/hr. ratio was 11. The culturing condition was the initial bacterial Moreover, in the method for producing extracellular pro cell concentration of about OD 0.03, an initial pH value tein from genus Tepidimonas of the present disclosure, the of 7.0, a stirring rate of 200 rpm, total volume of reaction initial pH of the culture medium is pH 5-pH 9, but it is not 10 was 4 L, and the culturing time was 80 hours. limited thereto. In one embodiment, the initial pH of the culture medium is about pH 7. In the foregoing method for producing extracellular pro TABLE 1. tein from genus Tepidimonas of the present disclosure, in Ingredients for the culturing medium fermenting, the final pH of the culture medium is about pH 15 4-pH 10, but it is not limited thereto. In one embodiment, the Ingredients Content (gL) final pH of the culture medium is about pH 8. Acetate 3.11 Furthermore, in the method for producing extracellular NaHCO 2.49 protein from genus Tepidimonas of the present disclosure, KH2PO 1...SO (NH4)2SO O.S3 the concentration of Tepidimonas for culturing is 0.1-40 V% Yeast extract 1.00 of the medium, for example, 5 V 96, but it is not limited MgCl2 O.10 thereto. Trace element solution 1 mLL In one embodiment, the culturing medium for the method (Ingredients shown below) of producing extracellular proteins from genus Tepidimonas, Trace element solution including a carbon source, a nitrogen source, a phosphate, a 25 ZnSO7H2O O.10 carbonate, a magnesium chloride (MgCl), a yeast extract MnCl4H2O O.O3 and trace elements, the carbon source which is acetate, the HBO O.30 CoCl26H2O O.20 nitrogen source which is ammonium sulfate (NH4)2SO), CuCl2.H2O O.O1 the carbonate which is sodium bicarbonate (NaHCO), the NiCl26H2O O.O2 content of the acetate may be about 0.1 g/L-15 g/L, the 30 Na-MoO2H2O O.O3 content of the ammonium sulfate may be about 0.1 g/L-15 g/L, the content of the phosphate may be about 0.1 g/L-30 FIG. 2 shows the protein yields and production rates of g/L, the content of the sodium bicarbonate may be about 0.1 culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at g/L-20 g/L, the content of the MgCl, may be about 0.01 different culturing temperatures. g/L-10 g/L, the content of the yeast extract may be about 0.2 35 g/L-5 g/L, and the content of the trace elements may be According to FIG. 2, the protein concentration and the about 0.01 g/L-1 g/L. protein yields at 50° C. were better than those at 45° C. and In the foregoing method for producing extracellular pro 55° C., respectively is 8.59 mg/L and 5.67 mg/L per day at tein from genus Tepidimonas of the present disclosure, 500 C. wherein the fermenting is conducted in a fermenter, wherein 40 Therefore, 50° C. was the optimum culturing temperature the concentration of Tepidimonas for culturing is 0.1-40 V% for producing extracellular proteins from Tepidimonas fon of the medium, the initial pH of the culture medium is pH ticaldi sp. nov. KCTC 12528BP. 5-pH 9, the final pH of the culture medium is about pH 4-pH 10, the stirring rate for the fermenting is about 50-500 rpm, Example 2 the temperature for the fermentation is about 40-80° C., the 45 air feeding type is circulating the culture medium by intro Air feeding rate effect on producing extracellular protein ducing the medium out from the fermenter and sending it of genus Tepidimonas KCTC 12528BP back into the fermenter and, during the circulation, a gas is Bacteria of Tepidimonas fonticaldi sp. nov. KCTC mixed into the culture medium before it is sent back, the 12528BP are slightly thermophilic bacteria. The Tepidimo circulation ratio is about 0.1-12 L/hr, the gas is air, and the 50 nas KCTC 12528BP was cultured at different air feeding air feeding rate for the fermenting is about 0.001-0.1 VVm. rates to determine a suitable air feeding rate for a 5 L Furthermore, in the embodiment mentioned above, cul fermenter. The testing air feeding rates were 0 VVm, 0.0125 turing a Tepidimonas into a culturing medium for fermen VVm, and 0.025 VVm. The air feeding type was directly tation to obtain a Suspension, the Suspension comprises feeding the air into the bottom of the culture medium, as extracellular proteins from genus Tepidimonas. 55 shown in FIG. 1A. The extracellular proteins can effectively be obtained The Tepidimonas KCTC 12528BP was cultured by the from the genus Tepidimonas by using the methods of the culture medium with the ingredients shown in Table 1. In the present embodiments. culturing medium, the concentration of acetate was 3.11 g/L. EXAMPLES 60 ammonium sulfate was 0.53 g/L, and the carbon-nitrogen weight ratio was 11. The culturing condition was the initial Example 1 bacterial cell concentration of about OD 0.03, an initial pH value of 7.0, a stirring rate of 200 rpm, total volume of Temperature effect on producing extracellular protein of reaction was 4 L, and the culturing time was 80 hours. genus Tepidimonas KCTC 12528BP 65 FIG. 3 shows the protein yields and production rates of The Tepidimonas KCTC 12528BP was cultured at differ culturing Tepidimonas fonticaldi sp. nov. KCTC 12528BP at ent respective temperatures to determine the most suitable different air feeding rates. US 9,580,738 B2 7 8 According to FIG. 3, the protein concentration and the pH value and rate of liquid circulation was investigated, for protein yields at 0.025 VVm were higher than 0 VVm and increased to protein concentration and protein production. 0.0125 VVm, respectively, at 21.77 mg/L and 10.90 mg/L per day. Example 4 Therefore, in the later examples, the air feeding rate was controlled at 0.025 VVm for feeding. The suitable final pH Final pH value effect on producing extracellular protein of value and liquid circulation rate for increasing protein Tepidimonas fonticaldi sp. nov. KCTC 12528BP concentration and protein production were also investigated. The pH value of the culturing medium will directly affect the bacterial growth, thereby affecting the protein concen Example 3 10 tration. From the previous experiments, it is known that the pH value of culture anaphase is higher than pH 8.5. The Air feeding type effect on producing extracellular protein Tepidimonas KCTC 12528BP was cultured at different of genus Tepidimonas KCTC 12528BP respective final pH values to determine a suitable final pH According to Example 2, it is known that directly feeding value for a 5 L fermenter. The testing final pH value of pH 15 7.5, pH 8, or pH 8.5. the air into the bottom of the culture medium caused a The Tepidimonas fonticaldi sp. nov. KCTC 12528BP was gas-lift phenomenon at the culture system. When gas-lift cultured with the culture medium ingredients shown in Table increased, it resulted the protein concentration and protein 1. In the culturing medium, the concentration of acetate was concentration decreased. The Tepidimonas KCTC 12528BP 3.11 g/L, ammonium sulfate was 0.53 g/L, and the carbon was cultured with different air feeding types to determine the nitrogen weight ratio was 11. The culturing condition was most suitable air feeding type for a 5 L fermenter. The testing the initial bacterial cell concentration of about OD 0.03, types were (a) directly feeding the air into the bottom of the an initial pH value of 7.0, a stirring rate of 200 rpm, a culture medium (referred to FIG. 1A), (b) circulating the culturing temperature of 50° C., a feeding rate of 0.025 VVm, culture medium by introducing the medium out from the a liquid circulation rate of 1.5 L/hr (the air feeding type (a) fermenter and sending it back into the fermenter and, during 25 or (b)), the total volume of reaction was 4 L, and the the circulation, the air is mixed into the culture medium culturing time was 250 hours. before it is sent back (referred to FIG. 1B), and (c) circu The results are shown in FIG. 5. It shows the protein lating the culture medium by introducing the medium out concentration and protein production rate of culturing Tepi from the fermenter and sending it back into the fermenter dimonas fonticaldi sp. nov. KCTC 12528BP at different final and feeding the air into the top of the fermenter (referred to 30 pH values. FIG. 1C). According to FIG. 5, the bacterial and protein concentra The Tepidimonas fonticaldi sp. nov. KCTC 12528BP was tion was improved. Thus, controlling the final pH value was cultured with the culture medium ingredients shown in Table an essential factor and it would directly affect protein 1. In the culturing medium, the concentration of acetate was production. When the final pH value was controlled at 8.0, 3.11 g/L, ammonium Sulfate was 0.53 g/L, and the carbon 35 the protein production concentration and protein production nitrogen weight ratio was 11. The culturing condition was rate are about 36.84 mg/L and 18.17 mg/L/day, respectively, the initial bacterial cell concentration of about ODoo 0.03, and better than pH 7.5 and pH 8.5. an initial pH value of 7.0, a stirring rate of 200 rpm, a Thus, in the later experiments, controlling the final pH culturing temperature of 50° C., a feeding rate of 0.025 VVm, value of the culturing medium at pH 8.0. total Volume of reaction was 4L, and the culturing time was 40 130 hours. Example 5 According to FIG. 4, it shows that the protein yields, production rates and protein yields of per gram of acetate at Rate of liquid circulation effect on secreting extracellular different air feeding types. protein of Tepidimonas fonticaldi sp. nov. KCTC 12528BP The protein production rate rises to about 11.45 mg/L/day 45 In one embodiment, culturing Tepidimonas fonticaldi sp. using the direct air feeding type (type a), which is the best nov. KCTC 12528BP was performed in a 5 L fermenter at of the three types. However, the direct air feeding type (type different liquid circulation rates for circulating the culture a) caused the gas-lift phenomenon and decreased the protein medium by introducing the medium out from the fermenter yields of per gram of acetate. and sending it back into the fermenter and, during the In other words, air feeding type (b) (circulating the culture 50 circulation, the air is mixed into the culture medium before medium by introducing the medium out from the fermenter it is sent back, to determine a Suitable rate of liquid circu and sending it back into the fermenter and, during the lation. The testing bacterial was cultured under liquid cir circulation, the air is mixed into the culture medium before culation rates of 1.5 L/hr, 3.0 L/hr or 6.0 L/hr. it is sent back) has a higher concentration of protein pro The Tepidimonas fonticaldi sp. nov. KCTC 12528BP was duction and protein yields of per gram of acetate than type 55 cultured using the culture medium ingredients shown in (a) and type (c). However, using the air feeding type (b) to Table 1. In the culturing medium, the concentration of culture Tepidimonas fonticaldi sp. nov. KCTC 12528BP, the acetate was 3.11 g/L, ammonium sulfate was 0.53 g/L, and protein production rate is slightly lower than using the air the carbon-nitrogen weight ratio was 11. The culturing feeding type (a), presuming the effect was medium liquid condition was the initial bacterial cell concentration of about circulation rates. 60 ODoo 0.03, an initial pH value of 7.0, a final pH values of The protein concentration and protein production rates in 8.0, a stirring rate of 200 rpm, a culturing temperature of 50° the air feeding type (c) are lower than the air feeding type (a) C., a feeding rate of 0.025 VVm, the total volume of the and (b) for culturing Tepidimonas fonticaldi sp. nov. KCTC reaction was 4 L, and the culturing time was 240 hours. 12528BP. FIG. 6 shows the protein concentration and protein pro According to measure result it is knows that concentration 65 duction rate of culturing Tepidimonas fonticaldi sp. nov. of protein and per gram of acetate of protein yields. In the KCTC 12528BP at different liquid circulation rates. The air later examples the air feeding type was (b), effect of the final feeding type involves circulating the culture medium by US 9,580,738 B2 10 introducing the medium out from the fermenter and sending a nitrogen source selected from a group consisting of it back into the fermenter and, during the circulation, a gas ammonium sulfate ((NH4)2SO), ammonium nitrate is mixed into the culture medium before it is sent back. (NH4NO), ammonium chloride (NHCl) and urea; According to FIG. 6, the liquid circulation rates of 1.5 a phosphate; L/hr and 3.0 L/hr could effectively improve protein produc a carbonate; tion concentration and protein production rates, except liq magnesium chloride (MgCl); uid circulation rates of 6.0 L/hr. Furthermore, when the a yeast extract; and liquid circulation rate was controlled at 3.0 L/hr, the protein trace elements; production concentration and protein production rate was collecting the Suspension, the Suspension comprising the higher than under a liquid circulation rate of 1.5 L/hr. The 10 protein concentration and protein production rate were about extracellular proteins from genus Tepidimonas, 36.43 mg/L and 25.14 mg/L. wherein the air feeding type is about 0.001-0.1 VVm air Thus, the liquid circulation rate of 3.0 L/hr in a 5 L feeding rate. fermenter could be considered for use in a 100 L fermenter. 2. The method of producing extracellular proteins from 15 genus Tepidimonas as claimed in claim 1, wherein the genus Example 6 Tepidimonas comprises Tepidimonas fonticaldi, Tepidimo nas ignava, Tepidimonas aquatic or Tepidimonas taiwan Different amounts of bacterial strain implanted into cul aSS. ture medium effect on Secreting extracellular protein of 3. The method of producing extracellular proteins from Tepidimonas fonticaldi sp. nov. KCTC 12528BP genus Tepidimonas as claimed in claim 1, wherein the In one embodiment, in culturing the different amounts of Tepidimonas is Tepidimonas fonticaldi sp. nov. Tepidimonas fonticaldi sp. nov. KCTC 12528BP in a 100 L 4. The method of producing extracellular proteins from fermenter. The testing amount of inoculation were 2.5 V%. genus Tepidimonas as claimed in claim 3, wherein the 5.0 V '%, or 10.0 V '%. (inoculation concentration of Tepi Tepidimonas fonticaldi is Tepidimonas fonticaldi sp. nov dimonas fonticaldi sp. nov. KCTC 12528BP is about 25 KCTC 12528BP. ODoo-1.5). 5. The method of producing extracellular proteins from The Tepidimonas fonticaldi sp. nov. KCTC 12528BP was genus Tepidimonas as claimed in claim 1, wherein the cultured using the culture medium ingredients shown in content of the carbon source is about 0.1 g/L-15 g/L, the Table 1. In the culturing medium, the concentration of content of the nitrogen Source is about 0.1 g/L-15 g/L, the acetate was 3.11 g/L, ammonium sulfate was 0.53 g/L, and 30 content of the phosphate is about 0.1 g/L-30 g/L, the content the carbon-nitrogen weight ratio was 11. The culturing of the carbonate is about 0.1 g/L-20 g/L, the content of the condition was the initial bacterial cell concentration of about MgCl, is about 0.01 g/L-10 g/L, the content of the yeast ODoo 0.03, an initial pH value of 7.0, a final pH value of extract is about 0.1 g/L-20 g/L, and the content of the trace 8.0, a stirring rate of 200 rpm, a culturing temperature of 50° elements is about 0.01 g/L-1 g/L. C., the liquid circulation rates of 3.0 L/hr (circulating the 35 6. The method of producing extracellular proteins from culture medium by introducing the medium out from the genus Tepidimonas as claimed in claim 1, wherein the fermenter and sending it back into the fermenter and, during carbon-nitrogen weight ratio of the carbon Source to the the circulation, the air is mixed into the culture medium nitrogen source is about 1-20 in the culture medium. before it is sent back), total volume of reaction was 80 L, and 7. The method of producing extracellular proteins from the culturing time was 225 hours. 40 genus Tepidimonas as claimed in claim 1, wherein the The results are shown as FIG. 7. It shows under different carbon source is acetate. amounts of inoculation into the culture medium, the con 8. The method of producing extracellular proteins from centration of the protein and protein production rate of genus Tepidimonas as claimed in claim 1, wherein the Tepidimonas fonticaldi sp. nov. KCTC 12528BP. nitrogen source is ammonium Sulfate ((NH4)2SO4). According to FIG. 7, it is known that when the bacterial 45 9. The method of producing extracellular proteins from strain was inoculation into the culture medium of 5 v '%, the genus Tepidimonas as claimed in claim 1, wherein the maximum mean of protein production rate of Tepidimonas phosphate comprises potassium dihydrogen phosphate fonticaldi sp. nov. KCTC 12528BP is the largest, and the (KHPO4), disodium hydrogen phosphate (NaHPO) or production of protein yields are constant. dipotassium phosphate (KHPO). Therefore, the 5 v '% implant amount could be used in a 50 10. The method of producing extracellular proteins from 100 L fermenter for protein production. genus Tepidimonas as claimed in claim 1, wherein the It will be apparent to those skilled in the art that various phosphate is dipotassium phosphate (KHPO). modifications and variations can be made to the disclosed 11. The method of producing extracellular proteins from embodiments. It is intended that the specification and genus Tepidimonas as claimed in claim 1, wherein the examples be considered as exemplary only, with a true scope 55 carbonate is sodium bicarbonate (NaHCO). of the disclosure being indicated by the following claims and 12. The method of producing extracellular proteins from their equivalents. genus Tepidimonas as claimed in claim 1, wherein the trace elements comprises Zinc (Zn), manganese (Mn), boron (B), What is claimed is: cobalt (Co), copper (Cu), nickel (Ni) and molybdenum 1. A method of producing extracellular proteins from 60 (Mo). genus Tepidimonas, comprising: 13. The method of producing extracellular proteins from fermenting a Tepidimonas in a fermenter with a culture genus Tepidimonas as claimed in claim 1, wherein the medium in an air feeding type to obtain a Suspension; fermenting step is in a stirring which the stirring rate is about and 50-500 rpm. wherein the culture medium comprises: 65 14. The method of producing extracellular proteins from a carbon Source which is an organic acid, selected from a genus Tepidimonas as claimed in claim 1, wherein the group consisting of acetate, lactate and butyrate; fermenting step is stirring at the rate of about 200 rpm. US 9,580,738 B2 11 12 15. The method of producing extracellular proteins from 29. The method of producing extracellular proteins from genus Tepidimonas as claimed in claim 1, wherein the genus Tepidimonas as claimed in claim 24, wherein the temperature of fermenting step is about 40-80° C. medium circulation rate is about 3 L/hr, and the air feeding 16. The method of producing extracellular proteins from rate for feeding is about 0.025 VVm. genus Tepidimonas as claimed in claim 1, wherein the 30. The method of producing extracellular proteins from temperature of fermenting step is about 50° C. genus Tepidimonas as claimed in claim 1, wherein an initial 17. The method of producing extracellular proteins from pH of the fermenting is about pH 5-pH 9. genus Tepidimonas as claimed in claim 1, wherein the air 31. The method of producing extracellular proteins from feeding of fermenting step is comprises an air. genus Tepidimonas as claimed in claim 1, wherein an initial 18. The method of producing extracellular proteins from 10 genus Tepidimonas as claimed in claim 1, wherein the pH of the fermenting is about pH 7. fermenting is conducted in a fermenter, the air feeding type 32. The method of producing extracellular proteins from comprises directly feeding the air into the bottom of the genus Tepidimonas as claimed in claim 1, wherein a final pH culture medium; circulating the culture medium by intro of the fermenting is about pH 4-pH 10. ducing the medium out from the fermenter and sending it 15 33. The method of producing extracellular proteins from back into the fermenter and, during the circulation, the air is genus Tepidimonas as claimed in claim 1, wherein an initial mixed into the culture medium before it is sent back; or pH of the fermenting is about pH 8. circulating the culture medium by introducing the medium 34. The method of producing extracellular proteins from out from the fermenter and sending it back into the fermenter genus Tepidimonas as claimed in claim 1, wherein an initial and feeding the air into the top of the fermenter. concentration of Tepidimonas for fermenting is 0.1-40 V '% 19. The method of producing extracellular proteins from of the culture medium. genus Tepidimonas as claimed in claim 1, wherein the 35. The method of producing extracellular proteins from fermenting is conducted in a fermenter, and the air feeding genus Tepidimonas as claimed in claim 1, wherein an initial type is directly feeding the air into the bottom of the culture concentration of Tepidimonas for fermenting is 5 V% of the medium. 25 culture medium. 20. The method of producing extracellular proteins from 36. The method of producing extracellular proteins from genus Tepidimonas as claimed in claim 19, wherein an air genus Tepidimonas as claimed in claim 5, wherein the feeding for the fermenting is air. carbon Source is acetate, the nitrogen Source is ammonium 21. The method of producing extracellular proteins from sulfate ((NHA)SO), and the carbonate is sodium bicarbon genus Tepidimonas as claimed in claim 20, wherein the air 30 feeding rate for the fermenting is about 0.001-0.1 VVm. ate (NaHCO). 22. The method of producing extracellular proteins from 37. The method of producing extracellular proteins from genus Tepidimonas as claimed in claim 20, wherein the air genus Tepidimonas as claimed in claim 36, wherein the feeding rate for the fermenting is about 0.025 VVm. content of the acetate is about 0.1 g/L-15 g/L, the content of 23. The method of producing extracellular proteins from 35 the ammonium sulfate is about 0.1 g/L-15 g/L, the content genus Tepidimonas as claimed in claim 1, wherein the of the phosphate is about 0.1 g/L-30 g/L, the content of the fermenting is conducted in a fermenter, and circulating the sodium bicarbonate is about 0.1 g/L-20 g/L, the content of culture medium by introducing the medium out from the the MgCl, is about 0.01 g/L-10 g/L, the content of the yeast fermenter and sending it back into the fermenter in which, extract is about 0.1 g/L-20 g/L, and the content of the trace during the circulation, the air is mixed into the culture 40 elements is about 0.01 g/L-1 g/L. medium before it is sent back. 38. The method of producing extracellular proteins from 24. The method of producing extracellular proteins from genus Tepidimonas as claimed in claim 37, wherein the genus Tepidimonas as claimed in claim 23, wherein the air fermenting is conducted in the fermenter, wherein an initial mixing for circulating is feeding air. concentration of Tepidimonas for fermenting is 0.1-40 V '% 25. The method of producing extracellular proteins from 45 of the medium, an initial pH of the fermenting about pH genus Tepidimonas as claimed in claim 23, wherein the 5-pH 9, the final pH of the fermenting about pH 4-pH 10, a medium circulation rate is about 0.1-12 L/hr. stirring rate for the fermenting is about 50-500 rpm, a 26. The method of producing extracellular proteins from temperature for the fermenting is about 40-80° C., and genus Tepidimonas as claimed in claim 23, wherein the wherein the fermenting is conducted in a fermenter, circu medium circulation rate is about 3 L/hr. 50 lating the culture medium by introducing the medium out 27. The method of producing extracellular proteins from from the fermenter and sending it back into the fermenter in genus Tepidimonas as claimed in claim 24, wherein the air which, during the circulation, the air is mixed into the feeding rate for the feeding is about 0.001-0.1 VVm. culture medium before it is sent back, and the air feeding rate 28. The method of producing extracellular proteins from about 0.001-0.1 vvm. genus Tepidimonas as claimed in claim 24, wherein the air 55 feeding rate for the feeding is about 0.025 VVm. k k k k k