Zhilong Xiu

Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol

Abstract1,3-Propanediol and 2,3-butanediol are two promising chemicals which have a wide range of applications and can be biologically produced. The separation of these diols from fermentation broth makes more than 50% of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced diols, with particular emphasis on 1,3-propoanediol. Previous studies on the separation of 1,3-propanediol primarily include evaporation, distillation, membrane filtration, pervaporation, ion exchange chromatography, liquid–liquid extraction, and reactive extraction. Main methods for the recovery of 2,3-butanediol include steam stripping, pervaporation, and solvent extraction. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. Perspectives for an improved downstream processing of biologically produced diols, especially 1,3-propanediol are discussed based on our own experience and recent work. It is argued that separation technologies such as aqueous two-phase extraction with short chain alcohols, pervaporation, reverse osmosis, and in situ extractive or pervaporative fermentations deserve more attention in the future.

Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition

Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. Copyright 1998 John Wiley & Sons, Inc.

A novel strategy for integrated utilization of Jerusalem artichoke stalk and tuber for production of 2,3-butanediol by Klebsiella pneumoniae

Jerusalem artichoke stalk and tuber can serve as a low cost feedstock for the production of 2,3-butanediol. However, like other lignocellulosic materials, the sugar concentration in the hydrolysate prepared from Jerusalem artichoke stalk is too low to be utilized effectively by microorganisms. In this paper a novel strategy was proposed to increase the sugar concentration by adding Jerusalem artichoke tuber into the hydrolysate of the stalk. The sugar was then biotransformed into high-valued 2,3-butanediol by Klebsiella pneumoniae. Fed-batch simultaneous saccharification and fermentation (SSF) was effectively performed and 901.2 mmol/l (80.5 g/l) target products (2,3-butanediol plus acetoin) was obtained in 68 h by a stage-shift aeration strategy. The concentration, yield and productivity of target products were 16.9%, 16.8% and 23.4%, respectively, higher than the best results obtained with SSF operated under constant aeration. This showed that adding tuber to the stalk hydrolysate was a useful strategy for increasing the production of 2,3-butanediol from Jerusalem artichoke via fermentation.

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system

A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively, and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w) ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation processes.

Microwave-assisted aqueous two-phase extraction of piceid, resveratrol and emodin from Polygonum cuspidatum by ethanol/ammonium sulphate systems.

Microwave-assisted, aqueous two-phase extraction was investigated to obtain effective constituents, including piceid, resveratrol and emodin in Polygonum cuspidatum. An aqueous two-phase system consisting of 25% (w/w) ethanol 21% (w/w) (NH4)2SO4 gave equal yields of piceid, and 1.1- and 1.9-times higher yields of resveratrol and emodin, respectively, than that achieved by microwave-assisted extraction and heat reflux extraction. Three-separate operations, extraction, clarification and concentration, are hereby integrated into a single step to get higher yields at lower cost. This is therefore a potentially useful method for the extraction and purification of target products.

Separation of bio-based chemicals from fermentation broths by salting-out extraction.

The possibility of creating a biorefinery using inexpensive biomass has attracted a great deal of attention, which is mainly focused on the improvement of strains and fermentation, whereas few resources have been spent on downstream processing. Bio‐based chemical downstream processing can become a bottleneck in industrial production because so many impurities are introduced into the fermentation broth. This review introduces a technique referred to as salting‐out extraction, which is based on the partition difference between chemicals in two phases consisting of salts and polymers or hydrophilic solvents, hydrophobic solvents, and amphipathic chemicals. The effects of solvents and salts on the formation of two phases were discussed, as was the use of this method to recover bio‐based chemicals. This review focused on the separation of hydrophilic chemicals (1,3‐propanediol, 2,3‐butanediol, acetoin, and lactic acid) from fermentation broths. Diols could be recovered at a high yield from fermentation broths without pretreatment especially with a hydrophilic solvent‐based system, whereas the recovery of organic acids was slightly lower. Most of the impurities (cells and proteins) were removed during the same step. Extractive fermentations were also used for polymer‐based aqueous two‐phase systems.

Comparative molecular dynamics simulations of histone deacetylase‐like protein: Binding modes and free energy analysis to hydroxamic acid inhibitors

Histone deacetylases (HDACs) play an important role in gene transcription, and inhibitors of HDACs can induce cell differentiation and suppress cell proliferation in tumor cells. Histone deacetylase1 (HDAC1) binds suberanilohydroxamic acid (SAHA) and 7‐phenyl‐2, 4, 6‐hepta‐trienoyl hydroxamic acid (CG‐1521) with moderately low affinity (ΔG = −8.6 and −7.8 kcal mol−1). The structurally related (E)‐2‐(3‐(3‐(hydroxyamino)‐3‐oxoprop‐1‐enyl)phenyl)‐N1,N3‐diphenylmalonamide (SK‐683), a Trichostatin A (TSA)‐like HDAC1 inhibitor, and TSA are bound to the HDAC1 with −12.3 and −10.3 kcal mol−1 of ΔG, higher binding free energies than SAHA and CG‐1521. Histone deacetylase‐like protein (HDLP), an HDAC homologue, shows a 35.2% sequence identity of HDLP and human HDAC1. Molecular dynamics simulation and the molecular mechanics/generalized‐Born surface area (MM‐GBSA) free energy calculations were applied to investigate the factors responsible for the relatively activity of these four inhibitors to HDLP. In addition, computational alanine scanning of the binding site residues was carried out to determine the contribution components from van der Waals, electrostatic interaction, nonpolar and polar energy of solvation as well as the effects of backbones and side‐chains with the MM‐GBSA method. MM‐GBSA methods reproduced the experimental relative affinities of the four inhibitors in good agreement (R2 = 0.996) between experimental and computed binding energies. The MM‐GBSA calculations showed that, the number of hydrogen bonds formed between the HDLP and inhibitors, which varied in the system studied, and electrostatic interactions determined the magnitude of the free energies for HDLP‐inhibitor interactions. The MM‐GBSA calculations revealed that the binding of HDLP to these four hydroxamic acid inhibitors is mainly driven by van der Waals/nonpolar interactions. This study can be a guide for the optimization of HDAC inhibitors and future design of new therapeutic agents for the treatment of cancer. Proteins 2008.

Parameters identification problem of the nonlinear dynamical system in microbial continuous cultures

In this paper, the nonlinear dynamic system of continuous glycerol fermentations to 1,3-propanediol by klebsiella pneumoniae is investigated. Considering big errors between the experimental results and computational values in the previous works, we establish the parameter identification model for the system. The properties of the solutions for the nonlinear dynamic system are discussed and the identifiability of the parameters is proved. Finally the feasible optimization algorithm is constructed to find the optimal parameters for the system.

Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography.

Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K₂HPO₄ aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01-73 L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS-PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1-2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61 μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation.

Theoretical analysis of effects of metabolic overflow and time delay on the performance and dynamic behavior of a two-stage fermentation process

The performance and dynamic behavior of a two-stage fermentation process are studied for continuous cultivation of microorganisms with metabolic overflow, the growth of which is subject to inhibitions of substrate and product. Metabolic overflow of product and its inhibition of cell growth lead to multiplicity, in which a steady state with higher biomass or product concentrations can be obtained by operating the bioreactor under certain conditions. For instance, a high density cell cultivation should be carried out by increasing the initial concentration of substrate in feed at a constant dilution rate. In contrast, a high yield of target product to substrate could be obtained by decreasing the initial concentration of substrate in feed. If a time delay between biomass formation and the change of the operation conditions is higher than a critical value, persistent periodical oscillations would occur. The critical time delay is dependent on the operating conditions, i.e. the initial substrate concentration and dilution rate. It is very large when the initial concentration of substrate is relatively low or high at a constant dilution rate. A two-stage fermentation process in terms of dynamic behavior is more efficient than one stage fermentation in steady state. The bioreactor performance, e.g. substrate consumption, product concentration, yield of product from substrate, and productivity, can be improved by using self-sustained oscillations in a system of two bioreactors in series.