Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs

Intestinal inflammation correlates well with the increased synthesis of nitric oxide (NO), which is attributed mainly to the up-regulation of inducible nitric oxide synthase (iNOS). We optimized the use of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) as inducers to stimulate NO synthesis in Caco-2 cells using a Taguchi design. The results indicated that IFN-γ was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-γ and PMA using an optimal mixture of 8000 U/ml IFN-γ and 0.1 μg/ml of PMA resulted in a synergistic induction of NO synthesis. Further experiments using a 5-factor/2-level factorial design including Caco-2 growth conditions such as cell passage, culture medium composition, cell seeding time and density, and stimulation time were also performed. Cell seeding and stimulation times significantly (P < 0.05) affected NO synthesis, whereas culture medium and seeding density did not appreciably affect NO synthesis in Caco-2 cells. Western blotting and RT-PCR findings confirmed that the optimal mixture of IFN-γ and PMA effectively up-regulated iNOS mRNA and protein. The induced NO, iNOS mRNA, and protein were all inhibited by the iNOS selective inhibitor, aminoguanidine (AG).     

Optimizing the spectrofluorimetric determination of cefdinir through a Taguchi experimental design approach

The aim of this work is to optimize a spectrofluorimetric method for the determination of cefdinir (CFN) using the Taguchi method. The proposed method is based on the oxidative coupling reaction of CFN and cerium(IV) sulfate. The quenching effect of CFN on the fluorescence of the produced cerous ions is measured at an emission wavelength (λ_em) of 358 nm after excitation (λ_ex) at 301 nm. The Taguchi orthogonal array L₉ (3⁴) was designed to determine the optimum reaction conditions. The results were analyzed using the signal‐to‐noise (S/N) ratio and analysis of variance (ANOVA). The optimal experimental conditions obtained from this study were 1 mL of 0.2% MBTH, 0.4 mL of 0.25% Ce(IV), a reaction time of 10 min and methanol as the diluting solvent. The calibration plot displayed a good linear relationship over a range of 0.5–10.0 µg/mL. The proposed method was successfully applied to the determination of CFN in bulk powder and pharmaceutical dosage forms. The results are in good agreement with those obtained using the comparison method. Finally, the Taguchi method provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

Film analysis of activated sludge microbial discs by the Taguchi method and grey relational analysis

A biofilm model with substrate inhibition is proposed for the activated sludge growing discs of rotating biological contactor (RBC); this model is different from the steady-state biofilm model based on the Monod assumption. Both deep and shallow types of biofilms are examined and discussed. The biofilm models based on both Monod and substrate inhibition (Haldane) assumptions are compared. In addition, the relationships between substrate utilization rate, biofilm thickness, and liquid phase substrate concentration are discussed. The influence order of the factors that affect the biofilm thickness is studied and discussed by combining the Taguchi method and grey relational analysis. In this work, a Taguchi orthogonal table is used to construct the series that is needed for grey relational analysis to determine the influence priority of the four parameters S _B , kX _f , K _s, and K _i .     

高浓度有机质驯化活性污泥对比产生氢率的影响

利用驯化的活性污泥降解蔗糖制取氢气,设计了间歇浓度梯度驯化方案,驯化后不同来源活性污泥的糖代谢速率和产氢速率均有显著提高,使初始含糖量为76～84g/L(实际约6 6kgCOD/m3 ·d)培养液的比产氢率超过了含糖4 0g/L的低浓度培养液的比产氢率而无抑制现象。其中啤酒厂厌氧活性污泥、啤酒厂排污处污泥及南京城市排水沟污泥的蔗糖消耗率均达到85 %以上;比产氢率分别达2 2 4、2 35和2 14molH2 /mol蔗糖。揭示了高浓度有机质的活性污泥间歇浓度梯度驯化过程中糖代谢速率和产氢能力的演变规律,其适用于相当多的驯化活性活泥降解高浓度有机废液,以提高单位产氢能力。     

Aerobic granular sludge formation for high strength agro-based wastewater treatment

The present study investigates the formation of aerobic granular sludge in sequencing batch reactor (SBR) fed with palm oil mill effluent (POME). Stable granules were observed in the reactor with diameters between 2.0 and 4.0 mm at a chemical oxygen demand (COD) loading rate of 2.5 kg COD m⁻³ d⁻¹. The biomass concentration was 7600 mg L⁻¹ while the sludge volume index (SVI) was 31.3 mL g SS⁻¹ indicating good biomass accumulation in the reactor and good settling properties of granular sludge, respectively. COD and ammonia removals were achieved at a maximum of 91.1% and 97.6%, respectively while color removal averaged at only 38%. This study provides insights on the development and the capabilities of aerobic granular sludge in POME treatment.     

Studies on the maximization of recombinant Helicobacter pylori neutrophil-activating protein production in Escherichia coli: application of Taguchi robust design and response surface methodology for process optimization

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a major antigen responsible for the generation of immune response in an infected individual. The cloning and expression of the gene corresponding to neutrophil-activating protein (NAP) were followed by process development for enhanced production and purification. The production process was developed in two parts. In the first part, some of the cultivation medium components (viz. carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulphate) were optimized using the Taguchi robust experimental design. The intracellular NAP production level after 24 h of cultivation was considered as the target function or the dependent variable. There was a 76.8% increase in the NAP production level. Using this optimal medium composition obtained in the first part, the temperature of cultivation and the pH of cultivation medium were optimized in the second part. The NAP production level at hour 30 of cultivation was considered as the target function or the dependent variable. The optimal values for these two independent variables were 37.2 °C and 6.3 respectively. At this combination of temperature and pH, the theoretical maximum NAP production level was 1280 mg l^–1. This optimal combination was verified experimentally and the NAP production level was found to be 1261 mg l^–1. The optimization of the cultivation conditions resulted in a 61.5% increase in NAP production level. About a 2.91-fold overall increase in NAP production level at hour 24 of cultivation was achieved through process optimization.     

Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast

This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester. Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor. An L₁₈ orthogonal array was used to design the experiments. The reaction yield and the product's optical purity were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality. Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality. The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study. Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.     

Improving the biotreatment of hydrocarbons-contaminated soils by addition of activated sludge taken from the wastewater treatment facilities of an oil refinery

Addition of activated sludge taken from the wastewater treatment facilities ofan oil refinery to a soil contaminated with oily sludge stimulated hydrocarbonbiodegradation in microcosms, bioreactors and biopile. Microcosms containing50 g of soil to which 0.07 % (w/w) of activated sludge was added presented ahigher degradation of alkanes (80 % vs 24 %) and polycyclic aromatic hydrocarbons(PAHs) (77 % vs 49 %) as compared to the one receiving only water, after 30days of incubation at room temperature. Addition of ammonium nitrate or sterilesludge filtrate instead of activated sludge resulted in a similar removal of PAHsbut not of alkanes suggesting that the nitrogen contained in the activated sludgeplays a major role in the degradation of PAHs while microorganisms of thesludge are active against alkanes. Addition of sludge also stimulated hydrocarbonbiodegradation in 10-kg bioreactors operated during 60 days and in a 50-m³ biopile operated during 126 days. This biopile treatment allowed the use of the soil for industrial purpose based on provincial regulation (``C' criteria). In contrast, the soil of the control biopile that received only water still exceeded C criteria for C₁₀–C₅₀ hydrocarbons, total PAHs, chrysene and benzo[a]anthracene.The stimulation effect of sludge was stronger on the 4-rings than on 2-rings PAHs.The soil of the biopile that received sludge was 4–5 times less toxic than the control. These results suggest that this particular type of activated sludge could be used to increase the efficiency of the treatment of hydrocarbon-contaminated soils in a biopile.     

Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge

A novel continuously stirred anaerobic bioreactor (CSABR) seeded with silicone-immobilized sludge was developed for high-rate fermentative H2 production using sucrose as the limiting substrate. The CSABR system was operated at a hydraulic retention time (HRT) of 0.5-6 h and an influent sucrose concentration of 10-40 g COD/L. With a high feeding sucrose concentration (i.e., 30-40 g COD/L) and a short HRT (0.5 h), the CSABR reactor produced H2 more efficiently with the highest volumetric rate (VH2) of 15 L/h/L (i.e., 14.7 mol/d/L) and an optimal yield of ca. 3.5 mol H2/mol sucrose. The maximum VH2 value obtained from this work is much higher than any other VH2 values ever documented. Formation of self-flocculated granular sludge occurred during operation at a short HRT. The granule formation is thought to play a pivotal role in the dramatic enhancement of H2 production rate, because it led to more efficient biomass retention. A high biomass concentration of up to 35.4 g VSS/L was achieved even though the reactor was operated at an extremely low HRT (i.e., 0.5 h). In addition to gaining high biomass concentrations, formation of granular sludge also triggered a transition in bacterial community structure, resulting in a nearly twofold increase in the specific H2 production rate. According to denatured-gradient-gel-electrophoresis analysis, operations at a progressively decreasing HRT resulted in a decrease in bacterial population diversity. The culture with the best H2 production performance (at HRT = 0.5 h and sucrose concentration = 30 g COD/L) was eventually dominated by a presumably excellent H2-producing bacterial species identified as Clostridium pasteurianum.     

污水处理厂污泥膨胀和污泥发泡的比较分析

活性污泥法由于操作简单、处理效果好被广泛应用于市政污水和工业废水的处理。污泥膨胀和污泥发泡现象影响二次沉淀池的泥水分离过程和生物反应池的微生物量稳定,严重困扰着污水处理厂的正常运行,被称为污水处理厂的"癌症"。本文从污泥膨胀和污泥发泡的定义及分类出发,全面地比较了表征污泥膨胀和污泥发泡的方法、引起污泥膨胀和污泥发泡的丝状细菌种类及控制污泥膨胀和污泥发泡方法的异同,并探讨了污泥膨胀和污泥发泡问题的未来研究方向和控制策略,期望能够为今后污泥膨胀和污泥发泡问题的研究和调控提供有价值的参考。     

Optimization of media composition for improving conversion of isoeugenol into vanillin with Pseudomonas sp. strain KOB10 using the Taguchi method

Abstract

The popular demand for natural food additives has resulted in a number of processes for producing natural vanillin. Although there are chemical procedures and plant sources for vanillin production, microbial bioconversions are being sought as a suitable ‘natural’ alternative. The present paper describes the conversion of isoeugenol to vanillin by a novel bacterial strain isolated from soil. The strain was identified as Pseudomonas sp. strain KOB10 based on morphological and physiochemical characteristics and its 16S rDNA gene sequence. We optimized medium composition for vanillin production using a Taguchi experimental design. Eight factors, i.e. isoeugenol, glycerol, tryptone, K₂HPO₄, KH₂PO₄, Cu²⁺, Mg²⁺ and Ca²⁺ concentrations, were selected and experiments based on an orthogonal array layout of L₁₈ (2² × 3⁶) were performed. Analysis of the experimental data using the Taguchi method indicated that Cu²⁺ and glycerol concentrations had the highest impact on isoeugenol conversion into vanillin at a substrate concentration of 0.9 g L^?1. Under the optimized conditions, growing cells of Pseudomonas sp. strain KOB10 produced 0.153 g vanillin L^?1 from 0.9 g isoeugenol L^?1, with a molar yield of 18.3% after incubation for 48 h. To improve the vanillin yield, the effect of other bioconversion parameters including time of isoeugenol addition, initial isoeugenol concentration and conversion time was studied; the results showed a maximum concentration of 3.14 g vanillin L^?1 after a total incubation time of 88 h with 15 g isoeugenol L^?1, which corresponded to a molar yield of 22.5%. Further standardization and optimization for vanillin production was challenging.     

Enzyme treatment to reduce solids and improve settling of sewage sludge

The effect of microbial enzymes in reducing the disposable solid content of sludge was investigated. A mixture of industrial cellulase, protease, and lipase, in equal proportion by weight, reduced total suspended solids (TSS) by 30–50% and improved settling of solids. An increase in solid reduction was observed with increasing enzyme concentration. The effect of combinations of enzyme treatments indicated that two-enzyme combinations of protease and cellulase produced better solid reduction than individual enzymes and that lipase further augmented this effect. Among the individual enzymes, protease produced a more settleable sludge as compared to cellulase and lipase. Adjustment of the pH of the enzymatically treated sludge to the acidic range (pH 2–4) further improved solid reduction, and adjustment to the alkaline range (pH 10–12) improved settleability. Journal of Industrial Microbiology & Biotechnology (2001) 26, 383–386. Received 01 November 2000/ Accepted in revised form 29 April 2001     

Efficient experimental design and analysis of real-time PCR assays

Real-time polymerase chain reaction (qPCR) is currently the standard for gene quantification studies and has been extensively used in large-scale basic and clinical research. The operational costs and technical errors can become a significant issue due to the large number of sample reactions. In this paper, we present an experimental design strategy and an analysis procedure that are more efficient requiring fewer sample reactions than the traditional approach. We verified mathematically and experimentally the new design on a well-characterized model, to evaluate the gene expression levels of CACNA1C and CACNA1G in hypertrophic ventricular myocytes induced by phenylephrine treatment.     

Application of alkaline treatment for sludge decrement and humic acid recovery

A new method was introduced to reduce waste activated sludge and extract humic acid for liquid fertilizer. Sludge was disintegrated with NaOH (0.4 g/g dry solid, 8 h) and then centrifuged to obtain the supernatant. The residual sludge was then dewatered, while the supernatant was used to extract humic acid with an ultrafiltration membrane. The results showed that the alkaline treatment dissolved more than half of the sludge organic matter, which was composed of 24% humic acid by mass. After the supernatant was concentrated 20 times using a membrane with a molecular weight cutoff of 1000, the retentate contained 94.5% of the dissolved organics and could be used to produce humic acid fertilizer. Additionally, only 26% of the NaOH was consumed and the residual NaOH in the permeate flux could be reused. Due to the removal of water and organics, the dewatered sludge could be reduced by 60% when compared to samples that did not receive the alkaline treatment.     

Recent advances in membrane bio-technologies for sludge reduction and treatment

This paper is designed to critically review the recent developments of membrane bio-technologies for sludge reduction and treatment by covering process fundamentals, performances (sludge reduction efficiency, membrane fouling, pollutant removal, etc.) and key operational parameters. The future perspectives of the hybrid membrane processes for sludge reduction and treatment are also discussed. For sludge reduction using membrane bioreactors (MBRs), literature review shows that biological maintenance metabolism, predation on bacteria, and uncoupling metabolism through using oxic-settling-anaerobic (OSA) process are promising ways that can be employed in full-scale applications. Development of control methods for worm proliferation is in great need of, and a good sludge reduction and MBR performance can be expected if worm growth is properly controlled. For lysis-cryptic sludge reduction method, improvement of oxidant dispersion and increase of the interaction with sludge cells can enhance the lysis efficiency. Green uncoupler development might be another research direction for uncoupling metabolism in MBRs. Aerobic hybrid membrane system can perform well for sludge thickening and digestion in small- and medium-sized wastewater treatment plants (WWTPs), and pilot-scale/full-scale applications have been reported. Anaerobic membrane digestion (AMD) process is a very competitive technology for sludge stabilization and digestion. Use of biogas recirculation for fouling control can be a powerful way to decrease the energy requirements for AMD process. Future research efforts should be dedicated to membrane preparation for high biomass applications, process optimization, and pilot-scale/full-scale tracking research in order to push forward the real and wide applications of the hybrid membrane systems for sludge minimization and treatment.