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

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(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 S 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 10 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 10 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 10 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.
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