膜生物膜法在水污染控制及资源回收中的研究进展

传统以达标排放为核心目标的废水处理工艺往往以高能耗、高物耗换取污染物削减,形成了"减排污染物、增排温室气体"的尴尬局面,并不符合可持续发展理念。作为一种新型的膜处理技术,膜生物膜法可利用无泡曝气的方式将气态电子供体(甲烷、氢气)或受体(氧气)提供给附着在膜表面的微生物,从而驱动水体中的污染物去除,并产生一些极具回收潜力的物质,最终实现污染物削减、节能减排及资源回收三大目标的有机整合。本文系统介绍了膜生物膜的传质过程及其去除污染物的微观机制,探讨了膜生物膜法在水处理资源回收方面的研究前景,梳理了膜生物膜反应器在水污染控制方面的实验研究和中试应用现状,并总结了膜生物膜法面临的挑战及发展趋势。     

Effects of pH profiles on nisin production in biofilm reactor

Apart from its widely accepted commercial applications as a food preservative, nisin emerges as a promising alternative in medical applications for bacterial infection in both humans and livestock. Improving nisin production through optimization of fermentation parameters would make nisin more cost-effective for various applications. Since nisin production by Lactococcus lactis NIZO 22186 was highly influenced by the pH profile employed during fermentation, three different pH profiles were evaluated in this study: (1) a constant pH profile at 6.8 (profile 1), (2) a constant pH profile with autoacidification at 4 h (profile 2), and (3) a stepwise pH profile with pH adjustment every 2 h (profile 3). The results demonstrated that the low-pH stress exerted during the first 4 h of fermentation in profile 3 detrimentally affected nisin production, resulting in a very low maximum nisin concentration (593 IU ml⁻¹). On the other hand, growth and lactic acid production were only slightly delayed, indicating that the loss in nisin production was not a result of lower growth or shifting of metabolic activity toward lactic acid production. Profile 2, in which pH was allowed to drop freely via autoacidification after 4 h of fermentation, was found to yield almost 1.9 times higher nisin (3,553 IU ml⁻¹) than profile 1 (1,898 IU ml⁻¹), possibly as a result of less adsorption of nisin onto producer cells. Therefore, a combination of constant pH and autoacidification period (profile 2) was recommended as the pH profile during nisin production in a biofilm reactor.     

乳链菌肽自身免疫基因nisI的表达对乳链菌肽产量的影响

【目的】通过基因工程手段增加乳链菌肽(nisin)自身免疫基因nisI在nisin产生菌Lactococcus lactisNZ9800/pHJ201中的表达水平,增强该菌对nisin的抗性,从而达到提高nisin产量的目的。【方法】将带有强组成型启动子P59的免疫基因nisI克隆到nisin表达质粒pHJ201上,将重组质粒引入L.lactis NZ9800中,使nisI基因过量表达,得到重组菌株L.lactis NZ9800/pHMI,并比较该重组菌株与对照菌株L.lactis NZ9800/pHJ201的生长曲线、对nisin的抗性水平、抑菌活性及nisin产量的差异。【结果】nisI的表达对重组菌的生长速度没有明显的影响,却能促使重组菌株对nisin的抗性水平提高25%、在发酵6h和8h时,nisin的产量分别提高32%和25%。【结论】增加乳链菌肽自身免疫基因nisI的表达可以提高产生菌对nisin的抗性,从而提高乳链菌肽产量。     

Evaluation of culture medium for nisin production in a repeated-batch biofilm reactor

In this study, a biofilm reactor with plastic composite support (PCS), made by high-temperature extrusion of agricultural products and polypropylene, was evaluated for nisin production using L. lactis strain NIZO 22186. The high-biomass density of the biofilm reactor was found to contribute to a significantly shorter lag time of nisin production relative to a suspended-cell reactor. In comparison to glucose (579 IU/mL), sucrose significantly increased the nisin production rate by 1.4-fold (1100 IU/mL). However, results revealed that high levels of sucrose (8% w/v) had a suppressing effect on nisin production and a stimulating effect on lactic acid production. A high concentration of MgSO4.7H2O at 0.04% (w/v) was found to reduce the nisin production, while concentrations of KH2PO4 of up to 3% (w/v) did not have any significant effect on growth or nisin production. The best of the tested complex media for nisin production using the PCS biofilm reactor consisted of 4% (w/v) sucrose, 0.02% (w/v) MgSO4.7H2O, and 0.1% (w/v) KH2PO4. Nisin production rate in the biofilm reactor was significantly increased by 3.8-fold (2208 IU/mL) when using the best complex medium tested.     

Effects of fed-batch fermentation and pH profiles on nisin production in suspended-cell and biofilm reactors

A biofilm reactor not only shortens the lag phase of nisin production, but also enhances nisin production when combined with an appropriate pH profile. Due to the substrate inhibition that takes place at high levels of carbon source, fed-batch fermentation was proposed as a better alternative for nisin production. In this study, the combined effects of fed-batch fermentation and various pH profiles on nisin production in a biofilm reactor were evaluated. The tested pH profiles include 1) a constant pH profile at 6.8 (profile 1), 2) a constant pH profile with an autoacidification after 4 h (profile 2), and 3) a step-wise pH profile with pH adjustment every 2 h (profile 3). When profile 1 was applied, fed-batch fermentation enhanced nisin production for both suspended-cell (4,188 IU ml⁻¹) and biofilm (4,314 IU ml⁻¹) reactors, yielded 1.8- and 2.3-fold higher nisin titer than their respective batch fermentation. On the other hand, pH profiles that include periods of autoacidification (profiles 2 and 3) resulted in a significantly lower nisin production in fed-batch fermentation (2,494 and 1,861 IU ml⁻¹ for biofilm reactor using profile 2 and 3, respectively) due to toxicity of excess lactic acid produced during the fermentation. Overall, this study suggested that fed-batch fermentation can be successfully used to enhance nisin production for both suspended-cell and biofilm reactors.     

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.     

The effect of nisin on murein synthesis

Nisin inhibits murein synthesis with concomitant accumulation of undecaprenyl-pyrophospho-MurNAc(pentapeptide) (lipid intermediate I). This inhibition is caused by the formation of a complex between the antibiotic and lipid intermediate I. Undecaprenyl-pyrophospho-MurNAc(pentapeptide)-GlcNAc (lipid intermediate II) also forms a complex with nisin. However, when murein synthesis is inhibited by nisin, this latter complex is not formed since lipid intermediate II is no longer synthesized.Abbreviations GlcNAc N-acetylglucosamine - MurNAc N-acetylmuramyl - Pentapeptide Ala--DGlu-Lys-DAla-DAla - C₅₅ undecaprenol Dedicated to Professor Otto Kandler on occasion of his 60th birthday     

Enhanced human lysozyme production in biofilm reactor by Kluyveromyces lactis K7

Lysozyme is an antimicrobial compound, which has been used in pharmaceutical and food industries. Chicken egg is the commercial source of lysozyme. However, human lysozyme is more effective and safer than egg-white lysozyme. Human milk is an important source for human lysozyme, but it is not feasible to provide the needed lysozyme commercially. Biofilm reactors provide passive immobilization of cells onto the solid support, which may lead to higher productivity. The aim was to evaluate the fermentation medium composition for enhanced human lysozyme production by Kluyveromyces lactis K7 in biofilm reactor with plastic composite supports. Yeast nitrogen base was selected as the best nitrogen source when compared to the yeast extract and corn steep liquor. Moreover, inhibition effect of NaCl and NH₄Cl at the concentrations of 25 and 50 mM was observed. Three factors Box–Behnken response surface design was conducted and the results suggested 16.3% lactose, 1.2% casamino acid, 0.8% yeast nitrogen base as optimum medium composition for maximum human lysozyme production. Overall, the human lysozyme production by K. lactis K7 was increased to 173 U/ml, which is about 23% improvement in biofilm reactor and 57% improvement compared to the suspended-cell fermentation.     

乳杆菌发酵上清对耐药白假丝酵母生物膜的作用

目的 研究德氏乳杆菌(Lactobacillus delbrueckii,L.delbrueckii)和发酵乳杆菌(Lactobacillus fermentum,L.fermentum)发酵上清液对3株耐氟康唑白假丝酵母临床分离株CA3、CA6、CA8生物膜形成和分散的作用。 方法 通过MIC试验,确认3株白假丝酵母临床分离株对氟康唑耐药;用96孔板构建体外白假丝酵母生物膜;用棋盘法分别检测L.delbrueckii、L.fermentum发酵上清液与氟康唑联用对3株白假丝酵母的作用;用XTT法对生物膜形成量进行定量分析;检测L.delbrueckii、L.fermentum发酵上清液与氟康唑联用对白假丝酵母时间-生长曲线的影响;显微镜拍照检测L.delbrueckii、L.fermentum发酵上清液单独和氟康唑联用对白假丝酵母生物膜形态的影响;平板培养法检测L.fermentum发酵上清液对白假丝酵母黏附作用的影响。 结果 CA3、CA6、CA8三株临床分离菌株对氟康唑耐药,MIC浓度均为8 μg/mL。L.delbrueckii与L.fermentum发酵上清液与氟康唑联用对3株耐药型白假丝酵母的生物膜形成与分散均未表现出协同作用,且发酵上清液与氟康唑联用效果不如发酵上清液单独处理效果好,L.fermentum发酵上清液对生物膜的分散作用较L.delbrueckii发酵上清液强。L.fermentum发酵上清液抑制白假丝酵母的初始黏附。 结论 L.delbrueckii、L.fermentum发酵上清液单独应用时均对耐氟康唑白假丝酵母生物膜的形成与分散有干预作用,与氟康唑联用时未表现出协同作用,抑制耐药白假丝酵母生物膜的作用可能与抑制菌丝形成和起始黏附有关。     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Recent advances for the production and recovery methods of lysozyme

Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.     

Developing microbial biofilm as a robust biocatalyst and its challenges

Biocatalysts, such as bacteria, yeast, fungi and the enzymes they produce, have been used for many industrial applications since they function as effective and environmentally friendly tools. Whole cells have also been used in many sophisticated bioprocesses since a number of sequential reactions can be catalyzed within the cells. However, the use of whole cells in suspension in batch, fed-batch and continuous processes has some limitations. For instance, the cultures are non-reusable, they are sometimes sensitive to the toxicity of substrates or products, there can be issues with short-term stability, and each of these issues can impede biocatalyst regeneration, perturbing the downstream process and causing complexity in running large scale continuous culture. Recently, biofilms have emerged as a new generation of biocatalysts to solve these limitations in the production of many bio-based materials, including chemicals, antibiotics, enzymes, bioethanol, biohydrogen, and electricity production via microbial fuel cells. The establishment of industrial processes using biofilms has the potential for high benefit in terms of low-cost cell immobilization without the necessity of added polymers or chemicals. Many small-scale biofilm reactors have been developed for the production of value-added products, and it may be challenging to establish it on an industrial scale.     

Bifurcational and dynamical analysis of a continuous biofilm reactor

A dynamical model of a continuous biofilm reactor is presented. The reactor consists of a three-phase internal loop airlift operated continuously with respect to the liquid and gaseous phases, and batchwise with respect to the immobilized cells. The model has been applied to the conversion of phenol by means of immobilized cells of Pseudomonas sp. OX1 whose metabolic activity was previously characterized (Viggiani, A., Olivieri, G., Siani, L., Di Donato, A., Marzocchella, A., Salatino, P., Barbieri, P., Galli, E., 2006. An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1. Journal of Biotechnology 123, 464-477). The model embodies the key processes relevant to the reactor performance, with a particular emphasis on the role of biofilm detachment promoted by the fluidized state. Results indicate that a finite loading of free cells establishes even under operating conditions that would promote wash out of the suspended biophase. The co-operative/competitive effects of free cells and immobilized biofilm result in rich bifurcational patterns of the steady state solutions of the governing equations, which have been investigated in the phase plane of the process parameters. Direct simulation under selected operating conditions confirms the importance of the dynamical equilibrium establishing between the immobilized and the suspended biophase and highlights the effect of the initial value of the biofilm loading on the dynamical pattern.     

Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production

The application of a cell immobilization technique to a biofilm-based photobioreactor was developed to enhance its photo-hydrogen production rate and light conversion efficiency. Rhodopseudomonas palustris CQK 01 was initially attached to the surface of packed glass beads to form a biofilm in this experiment. Then, the biofilm photobioreactor (BPBR) was illuminated by light-emitting diodes with light wavelengths of 470, 590 and 630 nm and hydrogen was evolved with glucose being the sole carbon source. Under the illumination condition of 5000 lux illumination intensity and 590 nm wavelength, the BPBR showed good hydrogen production performance: the hydrogen production rate was 38.9 ml/l/h and light conversion efficiency was 56%, while the hydrogen yield was 0.2 mol H₂/mol glucose. Furthermore, results show that the highest hydrogen production rate and glucose removal rate were obtained when the glucose concentration is 0.12 M, the optimal pH 7 and optimal temperature of influent liquid 25 °C.     

Enhancement of photo-hydrogen production in a biofilm photobioreactor using optical fiber with additional rough surface

In this study, a biofilm photobioreactor with optical fibers that have additional rough surface (OFBP-R) was developed and it was shown that additional rough surface greatly enhanced the biofilm formation and thus increased the cell concentration, leading to an improvement in the hydrogen production performance. The effects of operational conditions, including the influent substrate concentration, flow rate, temperature and influent medium pH, on the performance of OFBP-R were also investigated. The experimental results showed that the optimum operational conditions for hydrogen production were: the influent substrate concentration 60 mM, flow rate 30 mL/h, temperature 30 °C and influent medium pH 7. Under the optimal operation conditions discovered in this work, the OFBP-R yielded fairly good and stable long-term performance with hydrogen production rate of 1.75 mmol/L/h, light conversion efficiency of 9.3% and substrate degradation efficiency of 75%.     

Syngas fermentation of Clostridium carboxidivoran P7 in a hollow fiber membrane biofilm reactor: Evaluating the mass transfer coefficient and ethanol production performance

Gasification followed by syngas fermentation is a unique hybrid process for converting lignocellulosic biomass into fuels and chemicals. Current syngas fermentation faces several challenges with low gas–liquid mass transfer being one of the major bottlenecks. The aim of this work is to evaluate the performance of hollow fiber membrane biofilm reactor (HFM-BR) as a reactor configuration for syngas fermentation. The volumetric mass transfer coefficient (K_La) of the HFM-BR was determined at abiotic conditions within a wide range of gas velocity/flowrate passing through the hollow fiber lumen and liquid velocity/flowrate passing through the membrane module shell. The K_La values of the HFM-BR were higher than most reactor configurations such as stir tank reactors and bubble columns. A continuous syngas fermentation of Clostridium carboxidivorans P7 was implemented in the HFM-BR system at different operational conditions, including the syngas flow rate, liquid recirculation between the module and reservoir, and the dilution rate. It was found that the syngas fermentation performance such as syngas utilization efficiency, ethanol concentration and productivity, and ratio of ethanol to acetic acid depended not only on the mass transfer efficiency but also the characteristics of biofilm attached on the membrane module (biofouling or abrading of the biofilm). The HFM-BR results in a highest ethanol concentration of 23.93 g/L with an ethanol to acetic acid ratio of 4.79. Collectively, the research shows the HFM-BR is an efficient reactor system for syngas fermentation with high mass transfer.     

Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa

In cystic fibrosis individuals, chronic lung infections and hospital-acquired pneumonia are caused by Pseudomonas aeruginosa. P. aeruginosa generates siderophores such as pyoverdine (PVD) as iron uptake systems to cover its needs of iron ions for growth and infection. lasR quorum sensing (QS) gene has a crucial function in PVD production and biofilm generation in P. aeruginosa. Fifty isolates of P. aeruginosa were obtained from clinical specimens of sputum (collected from individuals suffering from pulmonary infections). Antibiotic sensitivity test was performed for 50P. aeruginosa isolates by using 10 different types of antibiotics. All isolates of P. aeruginosa showed resistance for all 10 using antibiotics in this study. Ten multidrug resistant isoloates of P. aeruginosa were selected for next tests. Virulence factors of ten multidrug resistant isolates of P. aeruginosa, such as biofilm generation, PVD production, and lasR gene were detected. From results, all 10P. aeruginosa isolates can produce biofilm, PVD, and contain lasR gene. The produced amplicon for the lasR gene was 725 bp. After mice injection by fresh and heated PVD produced by P. aeruginosa PS10 LC619328.2, the fresh PVD caused 100 % mortality within five days using 0.3 ml of its concentration (37.4 µM), while (15.3 µM) of heated PVD (toxoid) caused 50 % mortality.     

Influence of nisin hinge-region variants on lantibiotic immunity and resistance proteins

The rising existence of antimicrobial resistance, confirms the urgent need for new antimicrobial compounds. Lantibiotics are active in a low nanomolar range and represent good compound candidates. The lantibiotic nisin is well studied, thus it is a perfect origin for exploring novel lantibiotics via mutagenesis studies. However, some human pathogens like Streptococcus agalactiae COH1 already express resistance proteins against lantibiotics like nisin.This study presents three nisin variants with mutations in the hinge-region and determine their influence on both the growth inhibition as well as the pore-forming activity. Furthermore, we analyzed the effect of these mutants on the nisin immunity proteins NisI and NisFEG from Lactococcus lactis, as well as the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1.We identified the nisin variant ₂₀NMKIV₂₄ with an extended hinge-region, to be an excellent candidate for further studies to eventually overcome the lantibiotic resistance in human pathogens, since these proteins do not recognize this variant well.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.