Protective Effect of Immobilized Ammonia Oxidizers and Phenol‐degrading Bacteria on Nitrification in Ammonia– and Phenol‐containing Wastewater

Phenol present in wastewaters from various industries has an inhibitory effect on nitrification even at low concentrations. Hence, the biological treatment of wastewater containing both phenol and ammonia involves a series of treatment steps. It is difficult to achieve nitrification capability in an activated sludge system that contains phenol at concentrations above the inhibitory level. Batch treatment of wastewater containing various concentrations of phenol showed that the ammonia oxidation capability of suspended Nitrosomonas europaea cells, an ammonia oxidizer, was completely inhibited in the presence of more than 5.0 mg/L phenol. To protect the ammonia oxidizer from the inhibitory effect of phenol and to achieve ammonia oxidation capability in the wastewater containing phenol at concentrations above the inhibitory level, a simple bacterial consortium composed of an ammonia oxidizer (N. europaea) and a phenol‐degrading bacterial strain (Acinetobacter sp.) was used. Ammonia oxidation did not occur in the presence of phenol at concentrations above the inhibitory level when suspended or immobilized N. europaea and Acinetobacter sp. cells were used in batch treatment. Following the acclimatization of the immobilized cells, accumulation of nitrite was observed, even when the wastewater contained phenol at concentrations above the inhibitory level. These results showed that immobilization was effective in protecting N. europaea cells from the inhibitory effect of phenol present in the wastewater.     

Activated packed bed bioreactor for rapid nitrification in brackish water hatchery systems

A packed bed bioreactor (PBBR) was developed for rapid establishment of nitrification in brackish water hatchery systems in the tropics. The reactors were activated by immobilizing ammonia-oxidizing (AMONPCU-1) and nitrite-oxidizing (NIONPCU-1) bacterial consortia on polystyrene and low-density polyethylene beads, respectively. Fluorescence in situ hybridization demonstrated the presence of autotrophic nitrifiers belong to Nitrosococcus mobilis, lineage of β ammonia oxidizers and nitrite oxidizer Nitrobacter sp. in the consortia. The activated reactors upon integration to the hatchery system resulted in significant ammonia removal (P < 0.01) culminating to its undetectable levels. Consequently, a significantly higher percent survival of larvae was observed in the larval production systems. With spent water the reactors could establish nitrification with high percentage removal of ammonia (78%), nitrite (79%) and BOD (56%) within 7 days of initiation of the process. PBBR is configured in such a way to minimize the energy requirements for continuous operation by limiting the energy inputs to a single stage pumping of water and aeration to the aeration cells. The PBBR shall enable hatchery systems to operate under closed recirculating mode and pave the way for better water management in the aquaculture industry.     

Biosensor for the detection of bacteriophages based on a super-high-frequency resonator

Azospirillum lipoferum Sp59b microbial cells were immobilized on the surface of thin polystyrene films modified in plasma from a high-frequency discharge of argon (13.56 MHz). The optimal conditions for immobilization, under which cell activity was maintained with respect to specific bacteriophages, were established. It was shown that it is possible to record the interaction of immobilized microbial cells and bacteriophages with a microwave-based resonant system (5–8.5 GHz). It was found that the biosensor made it possible to distinguish the interaction of bacterial cells with specific bacteriophages from the control, in which such interaction was absent. With the obtained super-high-frequency sensor, it was possible to determine the content of ΦAl-Sp59b bacteriophages in a suspension containing ~106 phages/mL. The analysis time was about 10 min. The viability of microbial cells after immobilization was also determined with this sensor. The results obtained with the use of the sensor on the basis of a super-high-frequency resonator have shown that it is promising for the development of methods to determine viral particles and the viability of microbial cells.     

Modified hydroxyalkyl methacrylate gel as a support for immobilization of yeast cells

Summary Whole cells of Zygosaccharomyces lactis have been covalently linked to fine-grained hydroxyalkyl methacrylate gel Spheron P 1000 E which was prepared by treatment with epichlorhydrin and modified by an amine spacer. Experiments on the coupling of permeabilized and non-permeabilized cells to this gel support have shown that immobilized cell agregates may be obtained by the immobilization of thermally permeabilized cells. Cell clustering can be bypassed by immobilizing non-permeabilized cells. This immobilization procedure makes additional permeabilization possible.     

高效净水异养硝化细菌的筛选鉴定及固定化应用研究

本研究从海参养殖水体、泥土中筛选出4株具有硝化能力的异养硝化细菌。分别将其游离菌体细胞投入海参养殖水体,测定亚硝态氮、氨氮去除率,筛选出HS．NOB2为高效净化菌株,对HS-NOB2进行16SrDNA扩增及序列测定,初步鉴定为节杆菌（Arthrobactersp．）。利用海藻酸钠包埋法对高效净化菌体细胞进行固定化,将该固定化菌投入养殖水体及人工合成污水,研究其对水体中亚硝态氮、氨氮的处理效果,并与游离菌体细胞进行比较。结果表明,固定化后亚硝态氮去除率达到49．85％,氨氮去除率达到56．58％,均明显高于游离菌体细胞。上述研究为探寻水体净化提供了新思路,为水质改良剂的实际生产提供可选菌株。     

Biofilm formation and partial biodegradation of polystyrene by the actinomycete <Emphasis Type="Italic">Rhodococcus ruber</Emphasis>

Polystyrene, which is one of the most utilized thermoplastics, is highly durable and is considered to be non-biodegradable. Hence, polystyrene waste accumulates in the environment posing an increasing ecological threat. In a previous study we have isolated a biofilm-producing strain (C208) of the actinomycete Rhodococcus ruber that degraded polyethylene films. Formation of biofilm, by C208, improved the biodegradation of polyethylene. Consequently, the present study aimed at monitoring the kinetics of biofilm formation by C208 on polystyrene, determining the physiological activity of the biofilm and analyzing its capacity to degrade polystyrene. Quantification of the biofilm biomass was performed using a modified crystal violet (CV) staining or by monitoring the protein content in the biofilm. When cultured on polystyrene flakes, most of the bacterial cells adhered to the polystyrene surface within few hours, forming a biofilm. The growth of the on polystyrene showed a pattern similar to that of a planktonic culture. Furthermore, the respiration rate, of the biofilm, exhibited a pattern similar to that of the biofilm growth. In contrast, the respiration activity of the planktonic population showed a constant decline with time. Addition of mineral oil (0.005% w/v), but not non-ionic surfactants, increased the biofilm biomass. Extended incubation of the biofilm for up to 8 weeks resulted in a small reduction in the polystyrene weight (0.8% of gravimetric weight loss). This study demonstrates the high affinity of C208 to polystyrene which lead to biofilm formation and, presumably, induced partial biodegradation.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

微生态制剂对海水养殖系统硝化功能建立过程的影响

比较分析投加不同微生态制剂的海水养殖系统硝化功能建立的过程,为实际应用提供依据。利用海水素构建4个海水养殖系统,通过投加硝化细菌、光合细菌、枯草芽胞杆菌3种微生态制剂以及纤维毛球作为生物膜载体,比较分析不同养殖系统硝化功能的建立过程及硝化强度差异。投加硝化细菌+光合细菌和硝化细菌+枯草芽胞杆菌系统硝化功能建立时间分别为108 h和96 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.69 mg/(L·d)和1.36 mg/(L·d);添加纤维毛球的生物膜系统与生物絮团系统硝化功能建立时间分别为96 h和120 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.36 mg/(L·d)和0.98 mg/(L·d);投加碳源系统和对照系统硝化功能建立时间分别为84 h和96 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.18 mg/(L·d)和1.36 mg/(L·d)。硝化细菌+枯草芽胞杆菌系统硝化功能建立时间更短,但系统硝化强度低于硝化细菌+光合细菌系统;生物膜系统硝化强度高于生物絮团系统且硝化功能建立更快;添加碳源能够加快系统硝化功能建立过程,但降低了硝化细菌+枯草芽胞杆菌系统的硝化强度。     

固定化Ralstonia metallidurans CH34降解苯酚的研究

将既能耐抗重金属又能降解苯酚的细菌Ralstonia m etalliduransCH34固定化以提高其降酚效率。首先通过正交实验,得到了固定化该菌种的最优制备条件,然后对固定化细胞的降酚效果进行了研究。结果表明,固定化R.m etalliduransCH34的降酚效果明显优于游离细胞;抗重金属毒性方面也有较大提高;在加入额外碳源(甲苯,柠檬酸)情况下,固定化R.m etalliduransCH34进行苯酚降解时所受影响明显要小于游离态菌。     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

The effect of thiosulphate and other inhibitors of autotrophic nitrification on heterotrophic nitrifiers

It has been found that heterotrophic nitrification by Thiosphaera pantotropha can be inhibited by thiosulphate in batch and chemostat cultures. Allythiourea and nitrapyrin, both classically considered to be specific inhibitors of autotrophic nitrification, inhibited nitrification by Tsa. pantotropha in short-term experiments with resting cell suspensions. Hydroxylamine inhibited ammonia oxidation in chemostat cultures, but was itself fully oxidized. Thus the total nitrification rate for the culture remained the same.Heterotrophic nitrification by another organism, a strain of Pseudomonas denitrificans has also been shown to be inhibited by thiosulphate in short term experiments and in the chemostat. During these experiments it became evident that this strain is able to grow mixotrophically (with acetate) and autotrophically in a chemostat with thiosulphate as the energy source.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Microalgal cell immobilization for the long-term storage of the marine diatom Haslea ostrearia

To preserve the characteristics of the marine diatom Haslea ostrearia during long term storage, particularly size and shape, the algal cells were immobilized in alginate beads and stored at 4 ^∘C at reduced irradiance up to 4 months. Two clones of different size (Ho34, 63 μm and Ho40, 78 μm) were studied. With Ho34, a 10.4% decrease of the size was shown after 120 days, by using the conventional storage management, while it did not exceed 2.2% with immobilized cells. Consequently, H. ostrearia would have auxosporulated after 9 months compared to 52 months. At the same time, the rate of distortion (aberrant valve structure) free Ho34 cells reached 86% while no distorted immobilized cells were observed. Chorophyll content in cells showed that all the cells were alive up to 60 days and after this time cells immobilized in the core of the beads most probably suffered from the poor light diffusion. Culturability of the immobilized cells was tested immediately after their immobilization and after 60 and 120 days of storage. The delay (at least 5) before immobilized cells released from the beads decreased with the time of storage, because of the embrittlement of the beads during the storage. Once in fresh medium, the cells actively multiplied. We concluded that immobilization strongly slowed down the decrease in frustule size with time and allowed the storage of concentrated and calibrated inocula which could be inoculated directly in liquid culture medium without needing to dissolve the beads.     

Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles

An oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 mu, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100-mum aerobic layer.From the dynamics of the start-up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h(-1), and the maximum oxygen conversion rate was 1.0 mol Cmol(-1) h(-1). The maximum specific acetate uptake rate was 2.0 mol Cmol(-1) h(-1), and the Monod constant for acetate was 2.9 x 10(-2) mol m(-3). The maximum specific nitrification rate was 0.58 x 10(-1) mol Cmol(-1) h(-1), and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen-consuming, cells.     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Microbiological degradation of pentane by immobilized cells of <Emphasis Type="Italic">Arthrobacter sp.</Emphasis>

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42days of incubation at 20°C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85–95% pentane degradation during a 2month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

Purification of saliva agglutinin of<Emphasis Type="Italic"> Streptococcus intermedius</Emphasis> and its association with bacterial aggregation and adherence

Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.     

Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modified polyacrylonitrile membrane

Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrile membrane with glutaraldehyde as a coupling agent. The polymer carrier was preliminarily modified in an aqueous solution of NaOH and 1,2-diaminoethane. The content of amino groups was determined to be 0.58 mgeq g⁻¹. Two ways of immobilization were used—in the presence of 0.2 g l⁻¹ phenol and without phenol. The capability of two immobilized system to degrade phenol (concentration—0.5 g l⁻¹) as a sole carbon and energy source was investigated in batch experiments. Seven cycles of phenol biodegradation were conducted. Better results were obtained with the immobilized system prepared in the presence of phenol, regarding degradation time and phenol biodegradation rate. Scanning electron micrographs of the polyacrylonitrile membrane/immobilized Aspergillus awamori NRRL at the beginning of repeated batch cultivation and after the 7th cycle were compared. After the 7th cycle of cultivation the observations showed large groups of cells. The results from the batch experiments with immobilized system were compared to the results produced by the free strain. Phenol biodegradation experiments were carried out also in a bioreactor with spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation. 10 cycles of 0.5 g l⁻¹ phenol biodegradation were run consecutively to determine the degradation time and rate for each cycle. The design of the bioreactor appeared to be quite effective, providing large membrane surface to bind the strain.