Continuous ethanol production by immobilized yeast reactor

Summary Saccharomyces cerevisiae yeast immobilized in calcium alginate gel beads was employed in packed-bed column reactors for continuous ethanol production from glucose or cane molasses, and for beer fermentation from barley malt wort. With properly balanced nutrient content or periodical regeneration of cells by nutrient addition and aeration, ethanol production could be maintained for several months. About 7 percent (w/v) ethanol content could be easily maintained with cane molasses diluted to about 17.5 percent (w/v) of total reducing sugars at about 4 to 5 h residence time. Beer of up to 4.5 percent (wv) of ethanol could be produced from barley wort at about 2 h residence time without any addition of nutrients.     

Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemi l-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinia l-asparaginase is a tetrameric enzyme containing one site, Asn₂₈₁, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn₂₈₁ from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.     

Localization and production of novel l-asparaginase from Pectobacterium carotovorum MTCC 1428

Bacterial l-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free l-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of l-asparaginase was carried out using cell fractionation techniques. The activity of l-asparaginase was found to be 85 and 77% in the cytoplasm of P. carotovorum MTCC 1428 grown on medium containing l-asparagine and combination of l-asparagine and glucose respectively. Among the tested carbon sources, l-asparagine or the combination of l-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of l-asparaginase. The maximum production of l-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of l-asparagine and glucose respectively. Yeast extract, l-asparagine and peptone have shown significant effect on the production of l-asparaginase. P. carotovorum MTCC 1428 has assimilated l-asparagine as an essential carbon source for maximizing the production of l-asparaginase.     

粪便微生物宏基因组来源L-天冬酰胺酶的性质表征及应用研究

l-天冬酰胺酶是氨基酸代谢的关键酶,广泛应用于食品和医药领域,肠道菌群及其产生的l-天冬酰胺酶与宿主健康和疾病关系密切。【目的】获取肠道微生物来源的新型l-天冬酰胺酶,并对其进行性质表征和应用研究。【方法】以西黑冠长臂猿粪便微生物宏基因组为模板,克隆l-天冬酰胺酶基因,并在大肠杆菌中表达;对表达出的酶进行酶学性质研究,并用于处理薯条和癌细胞。【结果】克隆获得l-天冬酰胺酶基因NCasn5,全长996 bp,重组酶NCasn5分子量大小为37.296 kDa,最适pH为8.0,最适温度为60 ℃,K_m和V_max值分别为(3.33±0.21) mmol/L和(836.30±13.91)µmol/(min·mg),37 ℃体外血清半衰期约69 h。NCasn5能降低薯条中69.35%的丙烯酰胺含量,抑制人肝癌细胞QGY-7703和人恶性黑色素瘤细胞A-375细胞的生长。【结论】本研究获得的新型l-天冬酰胺酶,具有良好的热稳定性和较长的血清半衰期,不仅无谷氨酰胺酶活性,还能减少油炸薯条中丙烯酰胺的含量,也能诱导癌细胞QGY-7703和A-375凋亡,在食品加工及医药领域具有潜在的应用价值。     

A radiometric method for the measurement of l-asparaginase at concentrations below 1 × 10−5 I.U./ml: Technique and application

A radiometric method for the measurement of low levels of l-asparaginase activity (EC 3.5.1.1) has been devised. This technique uses a protracted incubation at 37°C to magnify amidohydrolytic activity. During this time, in most of the cases examined, l-[U-¹⁴C]asparagine is hydrolyzed to l-[U-¹⁴C]aspartic acid in a linear way; the l-[U-¹⁴C]aspartic acid so generated is transaminated with α-ketoglutaric acid by l-glutamic acid oxaloacetate transaminase (EC 2.6.1.1) and the [U-¹⁴C]oxaloacetic acid so formed is β-decarboxylated by Zn²⁺ at pH 5.0. Using this procedure, low levels of l-asparaginase have been detected in the serum of the chicken, horse, and ox. Use also has been made of maleimide, which inhibits mammalian l-asparaginase without affecting the enzymes from bacterial sources, to discount the possibility that bacterial contamination of mammalian samples was responsible for the activity seen. When a survey was conducted of the distribution of l-asparaginase in the organs of Mus musculus, testis was found to contain surprisingly high levels of the analogous hydrolase from liver. The applicability of this technique to the measurement of feeble activities of l-asparaginase leeched from Dacron tubing to which l-asparaginase has been covalently bound, also has been demonstrated.     

Scale-up and application of a cyclone reactor for fermentation processes

A cyclone reactor for microbial fermentation processes was developed with high oxygen transfer capabilities. Three geometrically similar cyclone reactors with 0.5?l, 2.5?l and 15?l liquid volume, respectively, were characterized with respect to oxygen mass transfer, mixing time and residence time distribution. Semi-empirically correlations for prediction of oxygen mass transfer and mixing times were identified for scale-up of cyclone reactors. A volumetric oxygen mass transfer coefficient k _L a of 1.0?s^?1 (available oxygen transfer rate with air: 29?kg?m^?3?h^?1) was achieved with the cyclone reactor at a volumetric power input of 40?kW?m^?3 and an aeration gas flow rate of 0.2?s^?1. Continuous methanol controlled production of formate dehydrogenase (FDH) with Candida boidinii in a 15?l cyclone reactor resulted in more than 100% improvement in dry cell mass concentration (64.5?g?l^?1) and in about 100% improvement in FDH space-time yield (300?U?l^?1?h^?1) compared to steady state results of a continuous stirred tank reactor.     

Continuous production of 4-ethylguaiacol by immobilized cells of salt-tolerant Candida versatilis in an airlift reactor

The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.     

A pilot-scale study of total volatile fatty acids production by anaerobic fermentation of sewage in fixed-bed and suspended biomass reactors

A comparative study of a fermentation process for total volatile fatty acids (TVFA) production using pilot-scale fixed-bed (FAS) and suspended biomass (FER) reactors in which similar operational conditions was carried out. The influence of the changes of ambient temperatures at fixed operational conditions was also studied. Oxidation–reduction potential (ORP) increased and effluent pH decreased as the hydraulic retention time (HRT) decreased, which was favourable for TVFA production. Equations describing the ORP and pH variations with the HRT were obtained. ORP variation with HRT for FAS and FER reactors followed a logarithmic function with a regression coefficient, R², equal to 0.98. The variations of pH with HRT followed polynomial functions with regression coefficients of 0.96 and 0.98 for FAS and FER reactors, respectively. Hydrolysis process increased with the experiment duration. At the beginning of the experiment, effluent soluble COD (SCOD) decreased with respect to the influent but further effluent SCOD increased showing higher values compared to the influent. Cold temperatures were more favourable than summer temperatures for the accumulation of TVFA at the liquid effluent. The FAS reactor was more effective in the production of TVFA than the FER reactor. The maximum yields of TVFA were obtained at an organic volumetric loading rate (B_V) of 1.9 g COD/l per day, corresponding to an HRT of 3.4 h, for both reactors. A maximum increase of ammonia and phosphorus was observed at the maximum value of HRT coinciding with an increase of pH and a decrease of ORP, as could be previously observed. The average P/SCOD ratio for the influent and effluent were 0.06 and 0.05, respectively, for FAS and FER reactors. The average Ammonia/SCOD ratio for the influent and effluent were 0.15 and 0.14, respectively. These results demonstrate that effluent quality was improved by the treatment employed in case a further process of nutrient removal is carried out.     

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of thereactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Immobilisation of whole bacterial cells for anaerobic biotransformations

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA_1/2 values (residence time corresponding to 50 % nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA_1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50 % nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA_1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA_1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively). Received: 12 August 1996 / Received revision: 14 November 1996 / Accepted: 15 November 1996     

Plasmid Transfer between Marine Bacteria in the Aqueous Phase and Biofilms in Reactor Microcosms

Plasmid transfer of broad-host-range plasmid RP1 from marine Vibrio sp. strain S14 to marine strain SW5 under optimum conditions on the surface of nutrient plates was improved 2 orders of magnitude by using the plasmid transfer process to select an SW5 recipient more efficient than the wild type in receiving and/or maintaining the plasmid. This recipient strain, SW5H, was used to form biofilms under flow conditions on the surfaces of glass beads in reactors. The S142(RP1) donor strain was introduced to the reactors after either 48 or 170 h of biofilm formation, and production of transconjugants in the aqueous phases and biofilms without selection pressure was assessed. Plasmid transfer to the recipient cells in the biofilm was detected for biofilms formed for 170 h but not in those formed for 48 h. The plasmid transfer frequency was significantly higher (P < 0.05) among cells attached to the bead surfaces in the biofilm than among cells in the aqueous phase.     

Evaluation and modeling of biochemical methane potential (BMP) of landfilled solid waste: A pilot scale study

The main goal of this study was to present a comparison of landfill performance with respect to solids decomposition. Biochemical methane potential (BMP) test was used to determine the initial and the remaining CH₄ potentials of solid wastes during 27 months of landfilling operation in two pilot scale landfill reactors. The initial methane potential of solid wastes filled to the reactors was around 0.347 L/CH₄/g dry waste, which decreased with operational time of landfill reactors to values of 0.117 and 0.154 L/CH₄/g dry waste for leachate recirculated (R1) and non-recirculated (R2) reactors, respectively. Results indicated that the average rate constant increased by 32% with leachate recirculation. Also, the performance of the system was modeled using the BMP data for the samples taken from reactors at varying operational times by MATLAB program. The first-order rate constants for R1 and R2 reactors were 0.01571 and 0.01195 1/d, respectively. The correlation between the model and the experimental parameters was more than 95%, showing the good fit of the model.     

Optimum design of a series of CSTRs performing reversible Michaelis-Menten kinetics: a rigorous mathematical study

The optimum design of a given number of CSTRs in series performing reversible Michaelis-Menten kinetics in the liquid phase assuming constant activity of the enzyme is studied. In this study, the presence of product in the feed stream to the first reactor, as well as the effect of the product intermediate concentrations in the downstream reactors on the reaction rate are investigated. For a given number of N CSTRs required to perform a certain degree of substrate conversion and under steady state operation and constant volumetric flow rate, the reactor optimization problem is posed as a constrained nonlinear programming problem (NLP). The reactor optimization is based on the minimum overall residence time (volume) of N reactors in series. When all the reactors in series operate isothermally, the constrained NLP is solved as an unconstrained NLP. And an analytical expression for the optimum overall residence time is obtained. Also, the necessary and sufficient conditions for the minimum overall residence time of N CSTRs are derived analytically. In the presence of product in the feed stream, the reversible Michaelis-Menten kinetics shows competitive product inhibition. And this is, because of the increase in the apparent rate constant K^' _m that results in a reduction of the overall reaction rate. The optimum total residence time is found to increase as the ratio (‚₀) of product to substrate concentrations in the feed stream increases. The isomerization of glucose to fructose, which follows a reversible Michaelis-Menten kinetics, is chosen as a model for the numerical examples.     

Attainable region analysis for continuous production of second generation bioethanol

Background

Despite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed.

Results

We obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate.

Conclusions

In this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

     

Effects of operating parameters on performances of nitrification and denitrification processes

Nitrification and denitrification of synthetic wastewater was studied by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter (packed column) for denitrification. A glucose solution was fed to the denitrification column to supply carbon source. Effects of important process variables such as sludge age, hydraulic residence time and feed ammonium concentration on system's performance were investigated. Effluent ammonium-nitrogen (NH4-N) concentration decreased with increasing sludge age and hydraulic residence time and remained constant for sludge age and hydraulic residence times greater than 12 d and 15 h, respectively. Feed ammonium-nitrogen concentration above 200 mg/l resulted in significant levels of NH4-N in the effluent at Š_c = 15 d and Š_H = 12 h in nitrification. Performance of denitrification stage was not satisfactory for feed NO₃-N concentrations above 150 mg N/l resulting in significant effluent NO₃-N levels at hydraulic residence time of Š_H = 6 h.     

Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.     

Prediction of removal efficiency of Lanaset Red G on walnut husk using artificial neural network model

An artificial neural network (ANN) model was used to predict removal efficiency of Lanaset Red (LR) G on walnut husk (WH). This adsorbent was characterized by FTIR-ATR. Effects of particle size, adsorbent dose, initial pH value, dye concentration, and contact time were investigated to optimize sorption process. Operating variables were used as the inputs to the constructed neural network to predict the dye uptake at any time as an output. Commonly used pseudo second-order model was fitted to the experimental data to compare with ANN model. According to error analyses and determination of coefficients, ANN was the more appropriate model to describe this sorption process. Results of ANN indicated that pH was the most efficient parameter (43%), followed by initial dye concentration (40%) for sorption of LR G on WH.     

Biofilm producing indigenous bacteria isolated from municipal sludge and their nutrient removal ability in moving bed biofilm reactor from the wastewater

In the present study, improved moving bed biofilm reactor (MBBR) was applied to enhance the nutrient removal ability of the municipal wastewater. A total of 18 indigenous bacterial isolates were screened from the sewage sludge sample and nitrate reductase, nitrite reductase and hydroxylamine oxidase was analyzed. The strains Pseudomonas aeruginosa NU1 and Acinetobacter calcoaceticus K12 produced 0.87 ± 0.05 U/mg and 0.52 ± 0.12 U/mg hydroxylamine oxidase, 1.023 ± 0.062 U/mg and 1.29 ± 0.07 U/mg nitrite reductase, and 0.789 ± 0.031 U/mg and 1.07 ± 0.13 U/mg nitrate reductase. Nitrogen and phosphate removal improved by the addition of nutrient sources and achieved > 80% removal rate. pH and temperature of the medium also affected nutrient removal and improved removal was achieved at optimum level (p < 0.05). MBBR was designed with R1 (aerobic), R2 and R3 (anoxic) reactors. MBBR reactors removed acceptable level phosphorus removal properties up to 7.2 ± 3.8%, 42.4 ± 4.6%, and 84.2 ± 13.1% in the R1, R2, R3 and R4 reactors, respectively. Denitrification rate showed linear relationship at increasing concentrations nitrogen content in the reactor and denitrification rate was 1.43 g NO₂-N /m²/day at 1.5 g NO₂-N /m²/day. Dehydrogenase activity was assayed in all reactors and maximum amount was detected in the aerobic biofilm reactor. Based on the present findings, MBBRs and the selected bacterial strains are useful for the degradation domestic wastewater with minimum working area.     

Effect of starch addition on the biological conversion and microbial community in a methanol-fed UASB reactor during long-term continuous operation

The effect of starch addition on the microbial composition and the biological conversion was investigated using two upflow anaerobic sludge bracket (UASB) reactors treating methanolic wastewater: one reactor was operated with starch addition, and another reactor was operated without starch addition. Approximately 300 days of operation were performed at 30 kg COD/m³/d, and then, the organic load of the reactors was gradually increased to 120 kg COD/m³/d. Successful operation was achieved at 30 kg COD/m³/d in both reactors; however, the methanol-fed reactor did not perform well at 120 kg COD/m³/d while the methanol-starch-fed reactor did. The granule analysis revealed the granule developed further only in the methanol-starch-fed reactor. The results of the microbial community analysis revealed more Methanosaeta cells were present in the methanol-starch-fed reactor, suggesting the degradation of starch produced acetate as an intermediate, which stimulated the growth of Methanosaeta cells responsible for the extension of granules.     

Continuous assays of L-asparaginase by coupling with glutamic dehydrogenase and by cationic glass electrode

Continuous assays of l-asparaginase by coupling with the glutamic dehydrogenase reaction and by cationic glass electrode are described. The procedures are about equally sensitive although the latter is simpler and a more direct assay. The two methods gave identical specific activity values for a purified preparation of Escherichia colil-asparaginase.