Mass Transfer, Gas Holdup, and Kinetic Models of Batch and Continuous Fermentation in a Novel Rectangular Dynamic Membrane Airlift Bioreactor

Compared with conventional cylinder airlift bioreactors(CCABs)that produce coarse bubbles,a novel rectangular dynamic membrane airlift bioreactor(RDMAB)developed in our lab produces fine bubbles to enhance the volumetric oxygen mass transfer coefficient(k_(L)a)and gas holdup,as well as improve the bioprocess in a bioreactor.In this study,we compared mass transfer,gas holdup,and batch and con-tinuous fermentation for RNA production in CCAB and RDMAB.In addition,unstructured kinetic models for microbial growth,substrate utilization,and RNA formation were established.In batch fermentation,biomass,RNA yield,and substrate utilization in the RDMAB were higher than those in the CCAB,which indicates that dynamic membrane aeration produced a high k_(L)a by fine bubbles;a higher k_(L)a is more bene-ficial to aerobic fermentation.The starting time of continuous fermentation in the RDMAB was 20 h ear-lier than that in the CCAB,which greatly improved the biological process.During continuous fermentation,maintaining the same dissolved oxygen level and a constant dilution rate,the biomass accumulation and RNA concentration in the RDMAB were 9.71% and 11.15% higher than those in the CCAB,respectively.Finally,the dilution rate of RDMAB was 16.7% higher than that of CCAB during con-tinuous fermentation while maintaining the same air aeration.In summary,RDMAB is more suitable for continuous fermentation processes.Developing new aeration and structural geometry in airlift bioreac-tors to enhance k_(L)a and gas holdup is becoming increasingly important to improve bioprocesses in a bioreactor.

Continuous Fermentation of a Prodigiosin-Producing <i>Serratia marcescens </i>Strain Isolated from Soil

A red-pigmented bacterial strain NS-17 was isolated from soil, and physiological and biochemical characteristics and 16S rDNA analysis established this organism as a strain of Serratia marcescens. The red pigment isolated from cells of NS-17 was identified as prodigiosin. By analyzing factors affecting the production of prodigiosin, modified medium and culture conditions were set up, and a continuous fermentation method was carried out to take advantage of the mass production of foam during fermentation. Continuous fermentation was achieved in the following conditions: 32℃, 1:1 ventilation ratio, and medium with a nutrient concentration 5-fold higher than that of the fermentation medium (with half the inorganic salt concentration) supplied as a feed medium at a flow rate of 8 mL/min. For the first time of continuous fermentation of Serratia marcescens coupling with foam flotation, a high yield of prodigiosin was obtained. After 56 h of culturing, the total harvest of cells was enhanced 2.33-fold compared to that of batch fermentation, the total amount of prodigiosin was enhanced 2.70-fold compared to that of batch fermentation, and cells and prodigiosin were concentrated in the efflux broth automatically.

Continuous Ethanol Production Through Fermentation with Glucose and Xylose by Immobilized Yeasts in Two Cascade Fixed Beds

Glucose and xylose were used as mixed carbon sources for ethanol production by a cascade immobi-lized-cell reactor.In the first fixed-bed reactor,immobilized S.cerevisiae was used to metabolize the glucoseanaerobically,and in the second fixed-bed column,the immobilized Pichia stipitis was applied to metabolize xy-lose aerobically.The effect of xylose on the glucose uptake by S.cerevisiae and the effect of ethanol on the xylosemetabolization by P.stipitis were studied.A mathematical model taking both tbe substrate and xylose inhibitioninto consideration was suggested for the description of the two-substrate fermentation process,and a total effec-tiveness factor η_s in the model was derived to take into account the mass transfer resistances of intra-particlesand liquid films.Calculated results are in fair agreement with the experimental data.

Theoretical Analysis of the Application of Immobilized Cells to the Continuous Fermentation Process

This paper theoretically studies the continuous immobilized cell (IMC) fer mentation system. A kinetic model of IMC is established, and the relational expressions between production rate, substrate concentration, biomass concentration and dilution rate in the IMC continuous stirred tank reactor (CSTR) are derived. These equations and some numerical calculations show that as compared with the free cell system the IMC system has many advantages: high production rate, steady operation, and being independent of the dilution rote. They also indicate that the diffusion of substrate is a constraint to the production of metablite.

Principles and Case Studies of Fed Batch Fermentation and Continuous Fermentation

Modern fermentation processes include a variety of fermentation methods,such as fed batch fermentation and continuous fermentation.This paper will focus on the principles and case studies of these two methods.Fed batch fermentation originates from fractionation fermentation with closed culture and adjustment of the pH value of the carbon source,to which the process of feeding the carbon source to the cell culture in a controlled manner has been added.This type of fermentation is more commonly used compared to the other.Continuous fermentation is also a closed fermentation system,which can operate without restrictions by continuous or intermittent addition of fresh nutrient media to the fermenter;however,it is susceptible to contamination by stray bacteria and metabolic inconvenience.

Production of Ammonium Lactate by Fed-batch Fermentation of Rhizopus oryzaefrom Corncob Hydrolysate

L-(+)-Lactic acid production from corncob hydrolysate as a cheap carbohydrate source by fed-batch fermentation of Rhizopus oryzaeHZS6 was studied. After 96 h of fermentation in a 5 L fermentor, the final concentration of ammonium L-(+)-lactate, average productivity(based on initial xylose concentration) and maximum dry cell weight were 132.4 g/L, 1.38 g/(L·h), and 8.9 g/L respectively. The optical purity of L-(+)-lactate was 98.8%.

Strategies for Optimizing Feed Rate of Fed-Batch Yeast Fermentation by Fuzzy-Neural Network

In this paper,a novel fuzzy neural network model,in which an adjustable fuzzy sub-space was designed by uniform design,has been established and used in fed-batch yeast fermentationas an example.A brand-new optimization sub-network with special structure has been built andgenetic algorithm,guaranteeing the optimization in overall space,is introduced for the feed rateoptimization.On the basis of the model network,the optimal substrate concentration and theoptimal amount of fed-batch at different periods have been studied,aided with the optimizationnetwork and the genetic algorithm separately.The above results can be used as a basis for theestablishment of a fuzzy neural network controller.

Fed-Batch Fermentation for Spinosad Production in an Improved Reactor

As a kind of aerobic bacteria, Saccharopolyspora spinosa exhibits a high demand for oxygen. In the fermentation process, the methods of increasing ventilation and improving agitation speed are usually adopted to achieve higher values of dissolved oxygen. These methods decrease the efficiency of spinosad biosynthesis.In this study, an improved reactor was designed to solve these problems. The exhaust gas reflux device, impellers,and baffles were improved. Furthermore, we established the kinetic models for the cell growth, substrate consumption and spinosad generation in batch fermentation process. The simulation results were in good agreement with the experimental data. Spinosad production reached 583.86 mg/L after employing the suitable feeding strategy by fed-batch fermentation in the improved reactor, whereas it was only 157.01 mg/L before optimization. The method described can provide insight to strengthen spinosad production and can be extended to the culturing process of filamentous aerobic bacteria.

Strain and process engineering toward continuous industrial fermentation

Most current biotechnology industries are based on batch or fed-batch fermentation processes,which often show low productivity and high production costs compared to chemical processes.To increase the economic competitiveness of biological processes,continuous fermentation technologies are being developed that offer significant advantages in comparison with batch/fed-batch fermentation processes,including:(1)removal of potential substrates and product inhibition,(2)prolonging the microbial exponential growth phase and enhancing productivity,and(3)avoiding repeated fermentation preparation and lowering operation and installation costs.However,several key challenges should be addressed for the industrial application of continuous fermentation processes,including(1)contamination of the fermentation system,(2)degeneration of strains,and(3)relatively low product titer.In this study,we reviewed and discussed metabolic engineering and synthetic biology strategies to address these issues.

The Pichia pastoris enzyme production platform:From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass

The demand for industrial enzymes is continually rising,fueled by the growing need to shift towards more sustainable industrial processes.However,making efficient enzyme production strains and identifying optimal enzyme expression conditions remains a challenge.Moreover,the production of the enzymes themselves comes with unavoidable impacts,e.g.,the need to utilize secondary feedstocks.Here,we take a more holistic view of bioprocess development and report an integrative approach that allows us to rapidly identify improved enzyme expression and secretion conditions and make use of cyanobacterial waste biomass as feed for supporting Pichia pastoris fermentation.We demonstrate these capabilities by producing a phytase secreted by P.pastoris that is grown on cyanobacterium hydrolysate and buffered glycerol-complex(BMGY)medium,with genetic expression conditions identified by high-throughput screening of a randomized se-cretion library.When our best-performing strain is grown in a fed-batch fermentation on BMGY,we reach over 7000 U/mL in three days.

Evaluation of ruminal methane and ammonia formation and microbiota composition as affected by supplements based on mixtures of tannins and essential oils using Rusitec

Background Dietary supplements based on tannin extracts or essential oil compounds(EOC)have been repeatedly reported as a promising feeding strategy to reduce the environmental impact of ruminant husbandry.A previous batch culture screening of various supplements identified selected mixtures with an enhanced potential to mitigate ruminal methane and ammonia formation.Among these,Q-2(named after quebracho extract and EOC blend 2,composed of carvacrol,thymol,and eugenol)and C-10(chestnut extract and EOC blend 10,consisting of oregano and thyme essential oils and limonene)have been investigated in detail in the present study with the semi-continuous rumen simulation technique(Rusitec)in three independent runs.For this purpose,Q-2 and C-10,dosed according to the previous study,were compared with a non-supplemented diet(negative control,NC)and with one supplemented with the commercial EOC-based Agolin^(R) Ruminant(positive control,PC).Results From d 5 to 10 of fermentation incubation liquid was collected and analysed for pH,ammonia,protozoa count,and gas composition.Feed residues were collected for the determination of ruminal degradability.On d 10,samples of incubation liquid were also characterised for bacterial,archaeal and fungal communities by high-throughput sequencing of 16S rRNA and 26S ribosomal large subunit gene amplicons.Regardless of the duration of the fermentation period,Q-2 and C-10 were similarly efficient as PC in mitigating either ammonia(-37%by Q-2,-34%by PC)or methane formation(-12%by C-10,-12%by PC).The PC was also responsible for lower feed degradability and bacterial and fungal richness,whereas Q-2 and C-10 effects,particularly on microbiome diversities,were limited compared to NC.Conclusions All additives showed the potential to mitigate methane or ammonia formation,or both,in vitro over a period of 10 d.However,several differences occurred between PC and Q-2/C-10,indicating different mechanisms of action.The pronounced defaunation caused by PC and its suggested consequences apparently determined at least part of the mitigant effects.Although the depressive effect on NDF degradability caused by Q-2 and C-10 might partially explain their mitigation properties,their mechanisms of action remain mostly to be elucidated.

L-酪氨酸连续发酵工艺研究

当前工业上用微生物发酵法生产L-酪氨酸的工艺中,基本都是前期向发酵罐内加入基础培养基,中后期再流加各类营养物质,虽然整个发酵过程中减少了取料和放料的操作,保证发酵中物料不被浪费,但是,随着流加物质的不断增加,发酵液体系不断扩大,该过程在发酵后期需要不断地人为调控发酵参数,而人为调控中,难以避免调控参数波动,并最终影响发酵结果。因此,实验采用高效连续发酵,该工艺可以大大提高菌体活力,并且有效延长产酸高峰期,为了优化L-酪氨酸的高密度连续发酵生产工艺,通过对大肠杆菌TYR-05发酵培养,考察了L-酪氨酸发酵过程并且分析了菌体量、产酸量、产酸效率、糖酸转化率的情况,确定L-酪氨酸高密度连续发酵过程中接种量、糖速率、糖耗量、底糖浓度等关键条件,以达到高密度连续发酵工艺控制条件优化。实验结果表明在5 L发酵罐中,选择30%的种子接种量,发酵起始时,底物氯化胆碱浓度为1 g/L并每4 h向罐内流加0.2 g/L的氯化胆碱,发酵至12 h时,底糖耗尽,开始以12 g/(L·h)的补糖速率向罐内提供葡萄糖,并在此时开始放液,放液速率为0.13 L/h,使得装液量恒定在20%左右,发酵25 h时开始流加复合营养液,发酵35 h时最高菌体OD_(600)达到65,产酸量为55.8 g/L,糖酸转化率为25.4%,为L-酪氨酸连续发酵工业化生产提供了重要参考。

吡咯喹啉醌连续发酵工艺初探

吡咯喹啉醌(pyrroloquinoline quinine, PQQ)的微生物发酵生产中,常用补料分批发酵法,但是其发酵前期菌体生长较慢,发酵后期产量下降较快,整个发酵周期的生产效率较低。为了提高PQQ的发酵生产效率,初步研究了使用生丝微菌(Hyphomicrobium sp.)进行单级连续发酵生产PQQ的工艺。以PQQ补料分批发酵时产率最高的时刻作为连续发酵的起点,分别进行了PQQ的恒浊法连续发酵和恒化法连续发酵的研究。实验结果表明,以0.008 7 h-1的稀释率进行PQQ恒浊法连续发酵更好,发酵液中的菌体浓度能够保持稳定且发酵产量较高。与补料分批发酵相比,恒浊法连续发酵可以提高PQQ的生产效率(80.7%),同时还提高了底物甲醇的转化率(36.5%),具有良好的工业应用前景。

不同包被缓释尿素的瘤胃缓释效果的评价

[目的]本文旨在通过体外批次发酵、瘤胃灌注和持续动态人工瘤胃模拟系统等不同方法评价包被缓释尿素的瘤胃缓释效果。[方法]体外发酵试验:选取1种普通尿素和4种商业包被缓释尿素,体外培养24 h,采集不同时间点(0、1、3、6、9、12、24 h)发酵液测定氨氮浓度,将筛选出缓释效果最好的缓释尿素用于进一步的研究。瘤胃灌注试验:12只体重(平均27.6 kg)相近的安装有瘤胃瘘管的育肥公湖羊,随机平均分为2组,分别向瘤胃中灌注剂量为日干物质采食量0.5%的普通尿素或日采食量0.55%的缓释尿素(等氮当量),灌注后不同时间点(0、1、3、6、9、12和24 h)采集瘤胃液,测定pH值和氨氮浓度。人工瘤胃试验:在基础日粮中分别添加3.5%的豆粕(普通日粮组)、0.5%的普通尿素(普通尿素组)和0.55%的缓释尿素(缓释尿素组),使用玉米补充普通尿素和缓释尿素组能量的缺乏;试验重复3次,每次持续7 d(4 d适应期和3 d采样期),采样期每天早上投料前后不同时间点采集发酵液和溢流液,测定发酵参数和微生物蛋白浓度。[结果]体外发酵试验:在发酵开始的9、12 h,缓释尿素A组的氨氮浓度显著低于普通尿素组和其余3个缓释尿素处理组(P<0.05)。体内瘤胃灌注试验:在灌注尿素后1 h,缓释尿素组瘤胃pH值和氨氮浓度显著低于普通尿素组(P<0.05),在灌注尿素后6 h,缓释尿素组的pH值显著高于普通尿素组(P<0.05)。人工瘤胃模拟试验:相比于其他2组,日粮添加缓释尿素显著提高了发酵液中丙酸和微生物蛋白的浓度(P<0.05);相比于普通日粮组,日粮添加缓释尿素显著降低了发酵液pH值(P<0.05)。[结论]不同包被缓释尿素的瘤胃缓释效果存在差异,体外发酵筛选出的缓释尿素A在动物瘤胃灌注条件下同样表现出较好的缓释效果。在人工瘤胃发酵试验中添加缓释尿素A能够改善瘤胃发酵,促进微生物蛋白的合成,但对生长性能和瘤胃菌群的影响仍需动物饲养试验做进一步的验证。

基于细胞特性的代谢反应工程技术创新及产业应用

生物制造的核心是细胞代谢反应,它受到胞外环境和细胞内在特性的调控。传统代谢反应过程主要依赖细胞外的宏观参数和经验模型来进行优化,但难以充分发挥细胞潜能。本文综述了以细胞特性来凝练代谢反应工程的核心问题和共性关键技术的研究进展,主要包括细胞物质与能量代谢、遗传与生长的时空调控以及细胞集群效应等细胞特性与代谢反应机制研究、工程技术创新和相关产业应用。基于细胞特性的代谢反应工程技术创新实现了我国1,6二磷酸果糖、酵母核苷酸和燃料乙醇等重大产品的工程技术水平提升和产业突破。这些成果都是在欧阳平凯院士的指导下完成的,以此文纪念欧阳平凯院士。

响应面法优化干清猪粪半干法连续厌氧发酵试验参数

为实现干清猪粪连续高效厌氧发酵,以干清猪粪为原料进行连续厌氧发酵试验,利用实验室自制连续厌氧发酵反应器探究水力滞留时间(HRT)、搅拌间隔与搅拌转速对干清猪粪连续厌氧发酵的影响,采用响应面分析法进行参数优化,得到最优参数组合:HRT 19 d、搅拌间隔2 h、搅拌转速30 r/min。经过干清猪粪连续厌氧发酵试验验证,其平均日产气量达23.48 L,VS降解率56.47%,平均容积产气率2.35 L/(L·d),能够实现干清猪粪连续厌氧发酵的高效稳定运行。因此,干清猪粪的无害化处理可采用半干法连续厌氧发酵运行,从而实现干清猪粪的资源化利用。

“发酵工程综合实验-以L-苯丙氨酸连续发酵实验为例”虚拟仿真课程的建设与探索

发酵工程是一门应用基础课程,包含大量工程实验和实际操作。但是,目前国内高等院校的发酵工程综合实验普遍存在诸多困难,如教学课时难以安排,真实发酵过程难以提供,存在安全风险和过程难以反复实施。因此,基于“以虚补实、虚实结合”的原则,笔者开发了“发酵工程综合实验-以L-苯丙氨酸连续发酵实验为例”虚拟仿真课程。该文介绍此虚拟仿真课程的教学内容、原理及操作,教学目标、实施方法和评价体系,以及其应用与效果。学生通过自主式、浸入式、情景式、互动式和反思式的学习方法,切身体验发酵场景、仪器设备和实验材料,并探索实验参数设置和技术选择对实验结果的影响。教学实践表明,该虚拟仿真课程能降低实验运行成本,提高学习效率,确保师生操作安全,提升学生对所学知识和技术的理解与应用,并有助于培养创新型复合型的新工科人才。

L-酪氨酸高密度流加发酵工艺研究

为实现L-酪氨酸的大规模生产,提高L-酪氨酸的产量和糖酸转化率,供试菌株为大肠杆菌TYR-05,通过单因素实验、正交实验、流加发酵优化实验,以菌体密度、L-酪氨酸产量、糖酸转化率及代谢副产物为指标,探究MgSO_(4)·7H_(2)O、磷酸二氢钾、谷氨酰胺、甲硫氨酸、维生素B_(12)、维生素H、磷酸吡哆醛对L-酪氨酸发酵的影响,最后确定适合L-酪氨酸发酵的培养基为MgSO_(4)·7H_(2)O_(2).0 g/L、KH_(2)PO_(4)2.5 g/L、维生素B_(12)2.0×10^(-3)g/L、PLP 1.0×10^(-3)g/L、维生素H 0.8×10^(-3)g/L,利用5 L发酵罐进行流加发酵生产,在初始发酵工艺的基础上,采用从10 h持续流加发酵的优化策略。实验结果表明,最高菌体密度达到65.0 g/L,L-酪氨酸产量为56.2 g/L,糖酸转化率为23.1%,乙酸作为副产物积累量减少到1.0 g/L,验证了L-酪氨酸高密度流加发酵策略的可行性,为L-酪氨酸及其他芳香族氨基酸的低成本、高效率工业化生产提供了一定的依据。