Production of bio-ethanol from soybean molasses by Saccharomyces cerevisiae at laboratory, pilot and industrial scales

The aim of this work was to develop an economical bioprocess to produce the bio-ethanol from soybean molasses at laboratory, pilot and industrial scales. A strain of Saccharomyces cerevisiae (LPB-SC) was selected and fermentation conditions were defined at the laboratory scale, which included the medium with soluble solids concentration of 30% (w/v), without pH adjustment or supplementation with the mineral sources. The kinetic parameters - ethanol productivity of 8.08g/Lh, Y(P/S) 45.4%, Y(X/S) 0.815%, m 0.27h(-1) and mu(X) 0.0189h(-1) - were determined in a bench scale bioreactor. Ethanol production yields after the scale-up were satisfactory, with small decreases from 169.8L at the laboratory scale to 163.6 and 162.7L of absolute ethanol per ton of dry molasses, obtained at pilot and industrial scales, respectively.     

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain

Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.     

Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales

The aim of this work was to research a bioprocess for bioethanol production from raw sweet potato by Saccharomyces cerevisiae at laboratory, pilot and industrial scales. The fermentation mode, inoculum size and pressure from different gases were determined in laboratory. The maximum ethanol concentration, average ethanol productivity rate and yield of ethanol after fermentation in laboratory scale (128.51 g/L, 4.76 g/L/h and 91.4%) were satisfactory with small decrease at pilot scale (109.06 g/L, 4.89 g/L/h and 91.24%) and industrial scale (97.94 g/L, 4.19 g/L/h and 91.27%). When scaled up, the viscosity caused resistance to fermentation parameters, 1.56 AUG/g (sweet potato mash) of xylanase decreased the viscosity from approximately 30000 to 500 cp. Overall, sweet potato is a attractive feedstock for be bioethanol production from both the economic standpoints and environmentally friendly.     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.     

Optimization of culture conditions and scale-up to plant scales for teicoplanin production by <Emphasis Type="Italic">Actinoplanes teichomyceticus</Emphasis>

This report describes the optimization of culture conditions for teicoplanin production by Actinoplanes teichomyceticus KCCM-10601, an identified high-teicoplanin-producing strain (US 2006/0134757 A1). Among the conditions tested, temperature, pH, and the dissolved oxygen tension (DOT) were key factors affecting teicoplanin production. When the temperature, pH, and DOT were controlled at 34 degrees C, 7.0 and 20-30%, respectively, a dry-cell weight of 42.8 g l(-1) and a teicoplanin production of 2.9 g l(-1) were obtained after 120 h of batch culture, corresponding to a specific teicoplanin content of 67.8 mg g-DCW(-1). Teicoplanin production was scaled-up from a laboratory scale (7-l fermenter) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V tip) as a scale-up parameter. Teicoplanin production at the laboratory scale was similar to those at the pilot and plant scales. This is the highest report of pilot- and plant-scale production of teicoplanin.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling

This article deals with the production by fermentation of a mycostatic and aromatic food additive based on propionic acid. Membrane bioreactors have been used from laboratory scale up to pilot and industrial production plants. Due to the high cell densities achieved by the sequential recycling mode of operation, a mixed acids solution was rapidly produced from whey permeate. The sterile fermented broth obtained was subsequently concentrated at different levels by evaporation and spray drying according to the projected use. Concentrated Propionibacterium cells (200 g . L(-1) DW) were obtained from the process by periodic bleeds and could be used to good effect as cheese starters, silage preservatives, or probiotics. Propionic acid concentrations from 30 to 40 g . L(-1) were easily achieved with no residual lactose. The highest volumetric productivity was 1.6 g . L(-1) . h(-1) for total acid and 1.2 g . L(-1) . h(-1) for propionic acid with a specific productivity of 0.035 h(-1). (c) 1993 John Wiley & Sons, Inc.     

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed‐batch processes, including at‐line sampling, deep freezer off‐line sample storage, and complete data handling. To enable scalable high‐throughput early‐stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom‐built hardware and software, fully automated milliliter‐scale microbial fed‐batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high‐throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early‐stage bioprocess development.     

Overproduction of L‐tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale‐up

L‐tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of E. coli was used to overproduce L‐tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimized anthranilic acid feeding into a dissolved oxygen‐stat (DO‐stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L‐tryptophan yield reached 0.211 g/g, 62.3% higher than the previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L‐tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale‐up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L‐tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess.

     

Real-time analyte monitoring of a fungal fermentation, at pilot scale, using in situ mid-infrared spectroscopy

Robust in situ biochemical monitoring is essential for the development of substrate feed control to optimize fermentation processes. The scale up of the fermentation for the fungus Glarea lozoyensis can benefit from such technology to improve the yield of the pharmaceutically important pneumocandin of interest and control the levels of unwanted analogues. A new in situ probe, using a diamond attenuated total reflection element, was evaluated at pilot scale for the quantitative measurement of fermentation analytes using Fourier transform mid-IR spectrometry. The new technology was shown to be stable, unaffected by reactor operation conditions of agitation, airflow, and backpressure, but sensitive to temperature control. Both glucose and phosphate were simultaneously monitored during a seed fermentation at 280 L pilot scale using complex medium with detection to 0.1 g/L for both analytes. Fructose, glutamate, and proline were monitored at 75 L scale using production media with detection limits of 0.1, 0.5, and 0.5 g/L respectively. Partial least squares calibration/prediction models were created for analytes of interest using off-line reference measurements and specific spectral regions. Good fits were obtained between off-line measurements and those predicted by in situ mid-IR. Standard errors of prediction (SEP) for glucose (range 18-0.1 g/L) and phosphate (range 11-7.5 g/L) were 0.16 and 1.8 g/L respectively with mean percentage errors (MPEs) around 2.5%. SEP values for the production process: fructose (range 20-0.1 g/L), glutamate (8-0.5 g/L), and proline (12-0.5 g/L) were 0.44, 0.6, and 0.5 g/L respectively with MPEs of 2.2, 5.3, and 10.1%. The technology effectively demonstrates quantitative multicomponent analysis of fermentation processes using in situ monitoring.     

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Abstract

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (k_La) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by Mut^S P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2?kW m^?3. Specific growth rate, and then dextranase production, increased as k_La and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2?L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut⁺ strain. After a scale-up process, at constant k_La, the specific growth rate was kept at 30?L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30?L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.     

Novel chemobiosynthetic approach for exclusive production of FK506

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess.     

In situ Raman spectroscopy for simultaneous monitoring of multiple process parameters in mammalian cell culture bioreactors

In this study, the application of Raman spectroscopy to the simultaneous quantitative determination of glucose, glutamine, lactate, ammonia, glutamate, total cell density (TCD), and viable cell density (VCD) in a CHO fed‐batch process was demonstrated in situ in 3 L and 15 L bioreactors. Spectral preprocessing and partial least squares (PLS) regression were used to correlate spectral data with off‐line reference data. Separate PLS calibration models were developed for each analyte at the 3 L laboratory bioreactor scale before assessing its transferability to the same bioprocess conducted at the 15 L pilot scale. PLS calibration models were successfully developed for all analytes bar VCD and transferred to the 15 L scale. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.     

Metabolic engineering for bioproduction of sugar alcohols

Sugar alcohols find applications in pharmaceuticals, oral and personal care products, and as intermediates in chemical synthesis. While industrial-scale production of these compounds has generally involved catalytic hydrogenation of sugars, microbial-based processes receive increasing attention. The past few years have seen a variety of interesting metabolic engineering efforts to improve the capabilities of bacteria and yeasts to overproduce xylitol, mannitol, and sorbitol. Examples include heterologous expression of yeast xylose reductase in Escherichia coli for the production of xylitol, coexpression of formate dehydrogenase, mannitol dehydrogenase, and a glucose facilitator protein in Corynebacterium glutamicum for mannitol production from fructose and formate, and overexpression of sorbitol-6-phosphate dehydrogenase in lactate dehydrogenase-deficient Lactobacillus plantarum to achieve nearly maximum theoretical yields of sorbitol from glucose.     

Butanol production from lignocellulosics

Clostridium spp. produce n-butanol in the acetone/butanol/ethanol process. For sustainable industrial scale butanol production, a number of obstacles need to be addressed including choice of feedstock, the low product yield, toxicity to production strain, multiple-end products and downstream processing of alcohol mixtures. This review describes the use of lignocellulosic feedstocks, bioprocess and metabolic engineering, downstream processing and catalytic refining of n-butanol.     

A simple high-yielding process for transient gene expression in CHO cells

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10? cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE.     

利用原生质体紫外诱变技术选育可常温发酵的中国被毛孢新菌株

中国被毛孢(Hirsutella sinensis)是天然冬虫夏草[Cordyceps sinensis(Berk)Sacc.]的真正无性型,其生长温度低(低于20℃),生长缓慢(发酵周期长达20～30d),对人工发酵的技术条件要求很高,因而大大增加了工业化发酵生产的成本和技术难度。为了解决上述问题,实验中从天然冬虫夏草子座中分离到中国被毛孢菌株BS-1,并对其进行了原生质体紫外诱变处理。经过粗筛和精筛,从中选出一株可常温下(25～28℃)发酵、生长迅速(发酵周期4～5 d)、有效成分(甘露醇和腺苷)含量显著高于原始菌株的诱变株BS-5962。通过连续10代PDA斜面继代培养和摇瓶试验,以及连续3批的1t罐的中试试验,检查结果表明所得诱变株性状优异而稳定。可为降低中国被毛孢的液体深层发酵成本和技术难度提供一种有效的解决方案。     

An ultra scale-down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges

Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra‐scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot‐scale disk‐type machines, was used in this work. Two recombinant P. pastoris strains, namely a X‐33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot‐ and a large‐scale disk‐type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X‐33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot‐scale runs during early stages of process development for therapeutic products. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1029–1036, 2012     

扩张柱床吸附层析回收纯化灌流培养生产的单克隆抗体

用扩张柱床吸附层析技术,一步回收纯化连续灌流培养的单克隆抗体。用Streamline SP阳离子交换介质在固定床柱XK16/20上进行条件摸索,扩张床柱Streamline25和50分别用于小规模条件优化和中试规模放大。培养液中的低浓度单抗经此步处理,浓缩10倍以上,纯度提高5～7倍,回收率＞90%,制备周期比固定柱床层析缩短一半以上。 根据培养液中单抗浓度的不同,一次处理量为18～50L,纯化规模由实验室水平（400mg）扩大至中试水平（2g）,生产成本和工艺复杂性大为降低。应用扩张柱床吸附层析技术,建立单克隆抗体回收纯化工艺,具有经济、简便、高效实用和良好的可放大性。