生物工程專業(yè)畢業(yè)設計英文翻譯

1、<p>　　High-Rate Continuous Production of Lactic Acid by Lactobacillus rhamnosus in a Two-Stage Membrane Cell-Recycle Bioreactor</p><p>　　Sunhoon Kwon, Ik-Keun Yoo, Woo Gi Lee, Ho Nam Chang, Yong Keun Ch

2、ang </p><p>　　Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, South Korea; E-m

3、ail: hnchang@kaist.ac.kr</p><p><b>　　Abstract</b></p><p>　　It is important to produce L(+)-lactic acid at the lowest cost possible for lactic acid to become a candidate monomer mater

4、ial for promising biodegradable polylactic acid. In an effort to develop a high-rate bioreactor that provides high productivity along with a high concentration of lactic acid, the performance of membrane cellrecycle bior

5、eactor (MCRB) was investigated via experimental studies and simulation optimization. Due to greatly increased cell density, high lactic acid productivity, 21</p><p>　　Keywords: Lactobacillus rhamnosus; lacti

6、c acid; high productivity; cell recycle; membrane bioreactor </p><p>　　INTRODUCTION</p><p>　　The efficiency of the membrane cell-recycle bioreactor (MCRB) was successfully demonstrated in a numb

7、er of previous studies of the high-volumetric productivity of lactic acid. With greatly increased density of biocatalysts, i.e., microbial cells, the volumetric productivity of lactic acid could go up to 160 g L?1 h?1 as

8、 reported in the study of Ohleyer et al. (1985), which is more than 20 times higher than that of the conventional batch and chemostat processes. </p><p>　　However, the high productivity is not the only requi

9、rement for the economic feasibility of the process. Timmer and Kromkamp (1994) found that the process might be primarily influenced by production capacity and product concentration and to a lesser extent by the volumetri

10、c productivity when annual lactic acid production capacity rose to as high as 4540 metric tons. In case lactic acid concentration is significantly low, the energy cost for water removal in the downstream process offsets

11、the bene</p><p>　　Thus, to enhance the economical advantage of the MCRB process, methods that increase the lactic acid concentration along with the high-cell density are required. Some authors, who considere

12、d this persistent problem of low-product concentration, conducted studies to obtain higher lactic acid concentration in MCRB. Xavier et al. (1995) reported a lactic acid concentration of 90 g/L with a productivity of 36

13、g L?1 h?1, while Tejayadi and Cheryan (1995) achieved 89 g/L and 22 g L?1 h?1 of lactic aci</p><p>　　A typical approach to overcome the above-mentioned problem, a low-product concentration due to severe prod

14、uct inhibition, is the use of a plug-flow reactor, which can be approximated by several continuous-stirred-tank receptors (CSTRs) in series (de Gooijer et al., 1996; Keller and Gerhardt, 1975; Luedeking and Piret, 1959b;

15、 Levenspiel, 1984). The advantages of the CSTRs-in-series against a single CSTR especially in lactic acid production were revealed by others in two- and three-stage CSTRs (Ae</p><p>　　In an effort to combine

16、 the advantage of both the bioreactor configurations—MCRB and multi-staged bioreactor— Kulozik et al. (1992) investigated the performance of a seven-staged cascade reactor with cell recycle. Cells in the outflow of the l

17、ast reactor were fivefold concentrated by a microfilter and recycled back to the first reactor. In comparison with a single-stage MCRB, the cascade reactor showed 4 times higher productivity, 28 g L?1 h?1, with complete

18、utilization of 100 g/L lactose, in wh</p><p>　　In this study, the performance of a new bioreactor configuration, two MCRBs in series, was investigated aiming at the highest volumetric productivity ever obtai

19、ned along with the lactic acid concentration as high as possible. Moreover, a simulation study was conducted to estimate the performance limit of MCRB with an unstructured kinetic model, which is validated by the experim

20、ent results.</p><p>　　MATERIALS AND METHODS</p><p>　　Microorganism and Culture Conditions</p><p>　　Lactobacillus rhamnosus (ATCC 10863), an obligatory anaerobic homofermentative L(+

21、)-lactic acid producer, was obtained from American Type Culture Collection (Rockville, MD). One-mL stock cultures were stored at ?76°C in Lactobacilli MRS medium (Difco, Detroit, MI) with 25%(v/v) glycerol. Precultu

22、res were prepared by transferring a stock culture to 200 mL of MRS medium and incubated at 42°C for 12 h and transferred to the main culture. The culture temperature was 42°C and the culture pH was contr</p&

23、gt;<p>　　Analytical Methods</p><p>　　Cell growth was measured by a spectrophotometer (Pharmacia Ultrospec 3000, Cambridge, UK) at a wavelength of 620 nm. Dry cell concentration was calculated from the

24、 optical density (OD620) with a linear correlation factor (one OD62040.32 g-dry cell weight per liter). Concentrations of lactic acid and glucose were determined by a high performance liquid chromatography (HPLC) system

25、equipped with a refractive-index detector (Hitachi L-6000, Tokyo, Japan). An HPLC column (Aminex 87H, Bio-Rad, Richmo</p><p>　　Membrane Cell-Recycle Bioreactor (MCRB) </p><p>　　In the experiment

26、s of a single-stage MCRB, a 400-mL water-jacketed glass reactor was employed, that was equipped with a hollow-fiber filtration unit UFP-100-H-4X2TCA (100 k NMWC, 0.065 m2 filtration area; A/G Technology Corporation, MA).

27、 A peristaltic pump, 07090-40 (Cole-Parmer, IL) was used to circulate the culture broth through the membrane unit with a flow rate of ca. 100 mL/min. For the two-stage operations, two identical MCRBs were serially connec

28、ted. Each MCRB consisted of a 1-L glass rea</p><p>　　Numerical Methods</p><p>　　The least squares regression was used to estimate the parameters of the fermentation kinetics. Numerical integrati

29、on to find steady-state values and constrained multivariable optimization to find the optimal operation variables were performed with the help of a software package, Matlab 5.0 (The Mathworks, Inc., USA). The constraints

30、 utilized in the optimization were the maximum cell density (Xm) and the maximum remaining glucose concentration (S).</p><p>　　DISCUSSION</p><p>　　To increase the bioreactor performance for the

31、production of lactic acid, a continuous lactic acid fermentation system coupled with membrane cell-separation technique (MCRB) has been studied. By greatly increased cell density in the reactor volumetric productivity co

32、uld be increased over 10times than the conventional batch and continuous fermentation. However, the concentration of lactic acid produced, a major factor for economic feasibility, could not be increased higher than 95 g/

33、L beyond whic</p><p>　　In conclusion, a systematic approach with MCRBs with multistaged operation can be carried out to predict optimal performances of lactic acid production, which experimentally proved tha

34、t two stage MCRBs can produce lactic acid in a high concentration with greatly increased volumetric productivity (type A).</p><p>　　References</p><p>　　[1] Aeschlimann A, Stasi LD, von Stockar U

35、. 1990. Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series. Enzyme Microb Technol 12:926–932.</p><p>　　[2] Amrane A, Prigent Y. 1999. Analysis of

36、growth and production coupling for batch cultures of Lactobacillus helveticus with the help of an unstructured model. Proc Biochem 34:1–10.</p><p>　　[3] Berry AR, Franco CMM, Zhang W, Middelberg APJ. 1999. G

37、rowth and lactic acid production in batch culture of Lactobacillus rhamnosus in a defined medium. Biotechnol Lett 21:163–167.</p><p>　　[4] Bibal B, Kapp C, Goma G, Pareilleux A. 1989. Continuous culture of S

38、treptococcus cremoris on lactose using various medium conditions. Appl Microbiol Biotechnol 32:155–159.</p><p>　　[5] Bibal B, Vayssier Y, Goma G, Pareilleux A. 1991. High concentration cultivation of Lactoco

39、ccus cremoris in a cell-recycle reactor. Biotechnol Bioeng 37:746–754.</p><p>　　[6] Bo¨rgardts P, Krischke W, Tro¨sch W, Brunner H. 1998. Integrated bioprocess for the simultaneous production of la

40、ctic acid and dairy sewage treatment. Bioprocess Eng 19:321–329.</p><p>　　[7] Bruno-Ba´rcena JM, Ragout AL, Cordoba PR, Sin?eriz F. 1999. Continuous production of L(+)-lactic acid by Lactobacillus casei

41、 in two-stage systems. Appl Microbiol Biotechnol 51:316–324.</p><p>　　[8] Cheryan M. 1998. Ultrafiltration and microfiltration handbook. Lancaster, PA: Technomic Publishing Company. 467 p.</p><p&g

42、t;　　[9] de Gooijer CD, Bakker WAM, Beeftink HH, Tramper J. 1996. Bioreactors in series: An overview of design procedures and practical applications. Enzyme Microb Technol 18:202–219.</p><p>　　[10] Dutta SK,

43、Mukherjee A, Chakraborty P. 1996. Effect of product inhibition on lactic acid fermentation: Simulation and modelling. Appl Microbiol Biotechnol 46:410–413.</p><p>　　[11] Gonc¸alves LMD, Xavier AMRB, Alm

44、eida JS, Carrondo MJT. 1991. Concomitant substrate and product inhibition kinetics in lactic acid production. Enzyme Microb Technol 13:314–319.</p><p>　　[12] Keller AK, Gerhardt P. 1975. Continuous lactic ac

45、id fermentation of whey to produce a ruminant feed supplement high in crude protein. Biotechnol Bioeng 17:997–1018.</p><p>　　[13] Kulozik U, Hammelehle B, Pfeifer J, Kessler HG. 1992. High reaction rate cont

46、inuous bioconversion process in a tubular reactor with narrow residence time distributions for the production of lactic acid. J Biotechnol 22:107–116.</p><p>　　[14] Kulozik U, Wilde J. 1999. Rapid lactic aci

47、d production at high cell concentrations in whey ultrafiltrate by Lactobacillus helveticus. Enzyme Microb Technol 24:297–302.</p><p>　　[15] Kwon S, Lee PC, Lee EG, Chang YK, Chang HN. 2000. Production of lac

48、tic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate. Enzyme Microb Technol 26:209–215.</p><p>　　[16] Levenspiel O. 1980. The Monod equation: A revisit and a generalization to pr

49、oduct inhibition situations. Biotechnol Bioeng 22:1671–1687. Levenspiel O. 1984. Chemical reaction engineering. New York: John Wiley & Sons. p 124–157.</p><p>　　[17] Litchfield JH. 1996. Microbiological

50、production of lactic acid. In: Neidleman SL, Laskin AI, editors. Advances in applied microbiology. Vol 42. San Diego: Academic Press. 69 p.</p><p>　　[18] Luedeking R, Piret EL. 1959a. A kinetic study of the

51、lactic acid fermentation. Batch process at controlled pH. J Biochem Microbiol Technol Eng 1:393–412.</p><p>　　[19] Luedeking R, Piret EL. 1959b. Transient and steady states in continuous fermentation. Theory

52、 and Experiment. J Biochem Microbiol Technol Eng 1:431–459.</p><p>　　[20] Major NC, Bull AT. 1985. Lactic acid productivity of a continuous culture of Lactobacillus delbrueckii. Biotechnol Lett 7:401–405.<

53、;/p><p>　　利用鼠李糖乳桿菌在兩級細胞膜循環(huán)</p><p>　　生物反應器中高速連續(xù)生產乳酸</p><p>　　——作者：Sunhoon Kwon，Yong Keun Chang</p><p>　　單位：韓國高等科學技術學院，化學與生物工程研究中心，E-mail: hnchang@kaist.ac.kr</p><

54、;p><b>　　摘　　要</b></p><p>　　眾所周知，乳酸是可生物降解材料聚乳酸的主要原料，所以找到以一種以最低的成本來生產L（+）－乳酸的方法具有非常重大的意義。為了找到一種可以高速地生產高濃度乳酸的生物反應器，我們對膜循環(huán)生物反應器（ MCRB ）的性能進行了研究，并進行了實驗仿真優(yōu)化。由于大大增加了細胞濃度，這個反應器的乳酸生產力可達到21.6gL- 1 h - 1。

55、但乳酸濃度卻不能超過83 g/L，當額外增加一個連續(xù)攪拌反應釜（ CSTR）附到MCRB旁邊時，可以大幅度的提高生產速率，乳酸濃度也可以提高到87 g / L，當兩個MCRBs串聯(lián)在一起時， 乳酸的生產力速率達到57gL- 1 h - 1，最終溶液中的乳酸濃度為92 g / L，這比以前所報道的使用葡萄糖基生產L （ + ）乳酸濃度超過85 g / L的最高的生產率還要高。此外，研究乳酸發(fā)酵動力學產生了以鼠李糖乳桿菌發(fā)酵生產乳酸為代表的

56、成功典范，該模型被認為是適用于大多數現(xiàn)有的MCRBs的數據 ，并且很好地吻合了Levenspiel的產品抑制模型，Luedeking-Piret產品形成動力學方程似乎是有效的代表發(fā)酵動力學。然而具有生產潛力的細胞（細胞密度相關參</p><p>　　© 2001 John Wiley ＆ Sons出版公司 生物 Bioeng 73 ： 25-34 ， 2001 。</p><p>

57、;　　關鍵詞：鼠李糖乳桿菌;乳酸;高生產力;細胞循環(huán);膜生物反應器</p><p><b>　　1　前　　言</b></p><p>　　膜細胞循環(huán)生物反應器（ MCRB ）的生產效率成功地證實了一些以往關于高容積生產乳酸的研究。Ohleyer等的研究報告指出，通過大量增加生物催化劑，即微生物細胞，乳酸的生產力可高達160 g L?1 h?1。 （ 1985年） ，這

58、比常規(guī)批次和恒化的生產工藝高出20倍以上。然而，高生產力并不是唯一的要求，這種工藝還必須在經濟上有可行性。Timmer and Kromkamp （ 1994年）發(fā)現(xiàn)，這一工藝可能主要受生產能力和產品的集中的影響，在較小程度時當年這種工藝生產乳酸的產能上升到高達4540噸。如乳酸濃度顯著低，能源成本中的水在去除抵消下游過程的好處，提高了生產力。從這個角度上講， MCRB有一個重要的問題有待解決：在乳酸濃度顯著低相比，間歇過程的乳酸濃度1

59、22 g / L的是容易實現(xiàn)的。此外，還有一份報告顯示以84 g L?1 h?1 的生產速率得到的D（+）L乳酸最終濃度為117 g/L （ Mehaia和Cheryan ， 1987年） ，而除部分MCRB工藝生產出的乳酸濃度低于90/g/L外所有其他的大多數生產的濃度低于60 g / L的（Cheryan ，1998年;里</p><p>　　因此，為加強MCRB工藝的經濟優(yōu)勢的方法有，隨著高密度的要求增加乳

60、酸濃度。一些考慮到這個長期存在的低濃度產品問題的作者對他進行了研究，并通過MCRB工藝獲得了較高濃度的乳酸。哈維爾等人。 （ 1995年）和Tejayadi和Cheryan （ 1995年）分別發(fā)表了以36 g L?1 h?1的生產速率得到濃度為90 g / L的乳酸和以22g L?1 h?1的生產速率得到濃度為89 g / L的乳酸的報道。</p><p>　　產品的濃度低是由于乳酸菌受到了嚴重抑制，這里有一個

61、很好的辦法來克服上述問題，我們可以通過使用推流反應器，它類似于很多連續(xù)化攪拌式受體（ CSTRs ）結合在一起（日Gooijer等。996年; Keller和戈哈德，1975年;Luedeking和Piret，1959年 ; Levenspiel ，1984年）。CSTRs的優(yōu)勢在一系列針對單一CSTR中特別是在乳酸生產中所揭示的其他兩個和三個階段CSTRs （艾緒里曼等人。1990年;布魯諾- Ba'rcena等。1999年;

62、根等。1991年）通過部分分離細胞的生長和乳酸生產階段提高乳酸的生產力和濃度，增加乳酸產量為代價的生物形成的后期;高純度的乳酸異構體長，L（ + ）乳酸菌通過增加新鮮細胞的數量;同時減少使用昂貴的養(yǎng)分——酵母膏。</p><p>　　為了結合雙方的優(yōu)勢，生物反應器的配置MCRB和多階段生物反應器Kulozik等。（1992）進行了一項七級聯(lián)反應器與細胞循環(huán)的研究。最后一個反應器中流出的細胞溶液通過收集器集中再生回

63、到第一座反應器中，相對于單級MCRB ，梯級反應器得到的生產率要高出4倍。達到 28克L- 1 h - 1，乳糖完整的利用率為100 g / L，其中的細胞濃度保持在20 g / L和的乳酸濃度約為72g/ L。</p><p>　　在這項研究中，對新型生物反應器的配置，即兩個MCRBs串聯(lián)的性能進行了研究，旨在在最高容積生產力的情況下不斷得到乳酸且其濃度盡可能高。此外，對估計MCRB的性能極限與非結構化的動力學

64、模型，進行了模擬研究，通過這個實驗驗證了結果。</p><p><b>　　2　材料與方法</b></p><p>　　2.1 微生物培養(yǎng)法及培養(yǎng)條件</p><p>　　鼠李糖乳桿菌（ ATCC 10863 ） ，一種同型發(fā)酵的具有極強的厭氧性的L （ + ）乳酸生產菌，它是從美國特種培養(yǎng)物保藏中心獲得的（位于美國馬里蘭州羅克維爾市） 。一毫

65、升庫乳桿菌菌種與（培養(yǎng)基，底特律， MI ）和的25 ％ （ V / V ）的甘油混合后在-76 ° C的條件下保存，Precultures準備通過在MRS培養(yǎng)基中，在42°C的條件下培養(yǎng)12小時，將菌株培養(yǎng)到200毫升，并轉移到主要化?？刂婆囵B(yǎng)溫度為42 ℃和通過使用氨水調節(jié)pH到6.0，MCRB工藝的培養(yǎng)基要有以下組成部分每升： 0.2Na3-Citrate·2H2O，1.0 g KH2PO4, 0.2

66、 g MgSO4·7H2O, 0.03 g MnSO4·H2O, 0.03 g FeSO4·7H2O, 和0.015mL硫酸。糖的濃度和酵母提取物將在結論中指出。除酵母提取物是單獨滅菌15分鐘外，所有培養(yǎng)基一起在121°C的條件下滅菌100分鐘。在結論中談到的培養(yǎng)體積包括循環(huán)流體培養(yǎng)基的體積。</p><p><b>　　2.2 分析方法</b><

67、;/p><p>　　細胞生長可通過分光光度計在波長為620納米時測定（法瑪西亞Ultrospec 3000 ，英國劍橋）一般可由干細胞濃度與光密度（OD620）的線性相關系數（1 OD62040.32克，干重每公升） 計算出來。乳酸的濃度和葡萄糖含量可由配備了折射率檢測器系統(tǒng)的高效液相色譜儀（HPLC）（日立L型6000 ，日本東京） 測定。HPLC柱使用時（ Aminex 87H ，酶標儀，里奇蒙， CA ）以0.

68、005M硫酸為流動相，在洗脫速度為0.6毫升/分鐘，而柱溫保持在50 ° C的濃度標準為1.0米乳酸（鹽，布克斯，瑞士）和10 g / L的葡萄糖（六西格瑪，圣路易斯，密蘇里州）用于高效液相色譜分析中。</p><p>　　2.3 膜細胞循環(huán)生物反應器（ MCRB ）</p><p>　　在單級MCRB的實驗中， 要應用到如下實驗器材：一；400毫升水套，它采用玻璃反應器并配備了

69、中空纖維超微粒過濾裝置- 100 - H的4X2TCA（100 k NMWC，0.065平方米過濾面積;阿/ g技術公司，馬）。二：蠕動泵， 07090-40型（科爾- Parmer ，白細胞介素）CA以100毫升/分鐘的速度推動發(fā)發(fā)酵液通過膜單元。 在兩個階段的行動，兩個相同的MCRBs是串行連接。每個MCRB包括一個1一L玻璃反應堆附有板和幀過濾單元， 一個Pellicon 2 BIOMAX 100V的（ 100 k NMWC ，

70、0.1平方米過濾面積，超純水，貝德福德，馬）與隔膜泵， 和一個P - 07090 -40 （科爾 Parmer ）的細胞再生裝置，其CA流速為600毫升/分鐘。 MCRB在接種前需要用含50 ％ （ V / V ）乙醇的無菌水徹底清洗。在操作過程中，需不斷向發(fā)酵罐中加入新的培養(yǎng)基同時排出產物。為了防止細胞密度去超過一定限度，造成過濾功能下降，需要從發(fā)酵罐中不斷抽出少量的發(fā)酵液 。在這兩個階段發(fā)酵過程中，從第一階段流出的發(fā)酵液用于第二階段

71、中。</p><p>　　2.4 數值分析方法</p><p>　　發(fā)酵動力學的參數可以用最小二乘回歸來估算。利用Matlab 5.0 （ MathWorks公司，公司，美國） 軟件進行數值積分找到穩(wěn)態(tài)值和約束多變量優(yōu)化以尋找到最佳操作變量。限制利用的優(yōu)化是最大的細胞密度（Xm）和最大其余血糖濃度（s） 。</p><p><b>　　討 論<

72、/b></p><p>　　為了提高生物反應器產乳酸的性能，我們對連續(xù)乳酸發(fā)酵系統(tǒng)加上膜細胞分離技術（ MCRB ）進行了研究。大大增加在固定體積發(fā)酵罐中的細胞密度，生產率比傳統(tǒng)的間歇和連續(xù)發(fā)酵提高了10倍以上。然而，乳酸實際生產中，最主要因素經濟上的可行性，此方法乳酸濃度高于95 g / L時，細胞的生長幾乎完全受到抑制。在初步單MCRB實驗中 ，即使在細胞密度保持在高于90 g / L時，得到的乳酸濃度

73、仍然很低，約51政/ L，（圖4 ）。當加入一個體積比MCRB大9的CSTR時，在放出細胞液之前如果讓它在在MCRB反應器中停留更長的時間， 則乳酸濃度會明顯提升，會達到87 g / L圖6 ） 。由此我們可以得出這樣的結論：在第二個反應器CSTR與另一MCRB 相連并且兩個階段的生物反應器與細胞循環(huán)同在這兩個階段前提下，可以高速生產高濃度的乳酸，如果使用兩個MCRBs系列，則可以以57 g L?1 h?1 的生產速率生產出濃度達到 9

74、2 g / L的乳酸（圖12 ） 。</p><p>　　最后，通過優(yōu)化多步驟MCRBs反應器，可以得到預期想得到的最優(yōu)生產乳酸的方法，實驗證明，利用兩階段MCRBs反應器可以高速生產高濃度的乳酸，從而使固定容積反應器的生產效率大大提高（ A型） 。</p><p><b>　　參考文獻</b></p><p>　　[1] Aeschliman

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