Harnessing the power of enzymes for environmental stewardship

Enzymes are versatile catalysts with a growing number of applications in biotechnology. Their properties render them also attractive for waste/pollutant treatment processes and their use might be advantageous over conventional treatments. This review highlights enzymes that are suitable for waste treatment, with a focus on cell-free applications or processes with extracellular and immobilized enzymes. Biological wastes are treated with hydrolases, primarily to degrade biological polymers in a pre-treatment step. Oxidoreductases and lyases are used to biotransform specific pollutants of various nature. Examples from pulp and paper, textile, food and beverage as well as water and chemical industries illustrate the state of the art of enzymatic pollution treatment. Research directions in enzyme technology and their importance for future development in environmental biotechnology are elaborated. Beside biological and biochemical approaches, i.e. enzyme prospection and the design of enzymes, the review also covers efforts in adjacent research fields such as insolubilization of enzymes, reactor design and the use of additives. The effectiveness of enzymatic processes, especially when combined with established technologies, is evident. However, only a limited number of enzymatic field applications exist. Factors like cost and stability of biocatalysts need to be addressed and the collaboration and exchange between academia and industry should be further strengthened to achieve the goal of sustainability.     

Performance of a three-stage aerobic RBC reactor in food canning wastewater treatment

Biological treatment using attached growth in a three-stage lab-scale rotating biological contactor (RBC) was implemented for wastewater from food cannery industries. The wastewater contained high level of organic compounds due to fish and fruit cleaning, cooking and filling processes. Nutrients available in the wastewater enhanced the growth of microorganisms and allowed the biological treatment to be effective. The RBC consisted of 54 parallel discs rotating in a reservoir and was arranged in three stages, i.e. 18 discs oriented in each stage. Effect of major operating and physical variables such as hydraulic retention time (HRT), disc submergence and disc rotational speed were examined in COD removal. For duration of 5 days, 96.4% BOD removal was achieved in batch experiment. BOD constant rate (k) and ultimate BOD were determined respectively, 0.8198 day⁻¹ and 6349 mg/l by Thomas graphical method. COD removal efficiency was increased from 85.3 to 97.4% while the HRT was increased from 24 to 48 h. The COD removal efficiency increased from 74.9 to 87.5% as the disc submergence was increased from 31 to 36%. At submergence level of 23.7%, removal efficiency was increased due to activation of second and third compartments. When the rotational speed was increased from 3 to 11 rpm, the COD removal efficiency was also increased from 62.7 to 93.7%, respectively. The stage COD removal efficiency was gradually decreased with an increase number of stage and about 88% of organic compounds were removed in the first stage of aerobic RBC, indicating that the single stage reactor may be sufficient in practical application.     

Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O₂-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O₂ was observed, starting already at > 700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O₂-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O₂-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme.     

Measurement of Oxygen Demand of Petroleum Hydrocarbon-Contaminated Groundwater

The standard biological oxygen demand (BOD) test was modified for application to petroleum hydrocarbon-contaminated groundwater. The goal was to assess the potential oxygen demand of plume constituents as part of a field trial investigating oxygen-enhanced in situ bioremediation. Modifications to standard BOD protocol included the use of an adapted microbial population developed from site groundwater and methods to minimize both the loss of volatile compounds and the exposure of samples to air. Results from this study indicated that the measured oxygen demand was significantly greater than the oxygen demand estimated solely by stoichiometric calculations from the concentrations of the analytes of typical regulatory concern, that is, benzene, toluene, ethylbenzene, and xylenes (BTEX). This is not surprising, because the petroleum hydrocarbon sources typically contain many organic contaminants other than BTEX, as well as potentially oxidizable natural dissolved organic matter and inorganic species typically present in hydrocarbon plumes. However, in practice, estimation of the total oxygen demand of a contaminated groundwater by exhaustive analyses of all oxidizable or aerobically degradable species typically will be infeasible. The modified BOD test may be a simple, low-cost, useful tool when assessing the potential for natural attenuation by aerobic biodegradation or designing methods to supply oxygen for enhanced aerobic bioremediation.     

Prediction of biodegradability for selected organic chemicals

Summary The 5-day BODs of 45 organic chemicals were determined using acclimated mixed microbial cultures. These chemicals included alcohols, acids, esters, ketones, aromatics and miscellaneous compounds. The BOD data were correlated with (1) water solubilities, (2) log of 1-octanol/water partition coefficients, (3) molar refractivities and volumes, (4) melting (m.p.) and boiling points, (5) number of carbon (C No.), hydrogen and oxygen atoms, (6) molecular weights, and (7) theoretical (Th) BODs of chemicals. Linear and secondorder polynomial regression analyses were used; the latter was also attempted with two or more independent variables. All prediction equations were compared for statistical merits. The equations, one from each regression type, with the highest prediction power were: log 5-day mmol BOD/mmol chemical=(1)–0.183+0.813 (log ThBOD), (2)–0.391+1.560 (log ThBOD) –0.532 (log ThBOD)², and (3) –0.4060+0.2470 (C No.) –0.0133 (C No.)²–0.0005 (m.p.). The measured BOD data for 43 additional chemicals were compared with the predicted values calculated through the above equations. The three equations predicted the BODs for 84–88% of the test chemicals within 80% of the experimental values. The mean percent relative standard deviations between predicted and experimental BOD values were statistically compared for these equations, and no significant difference (P0.01) in their predictive utility was found. The acclimation potential of an autochthonous microbial community cannot yet be predicted, but this study demonstrates that the process of active biodegradation for structurally dissimilar chemicals appears to correlate quantitatively with certain physicochemical parameters.     

Changes in water quality in Lake Päijänne following decrease of effluent load from the pulp and paper industry in 1969–1989

Lake Päijänne, the second largest lake in Finland, has been seriously polluted since the 1960s due to the effluents from pulp and paper mills, notably the Äänekoski sulphate and sulphite pulp mills situated about 50 km north of the lake, and the sulphite pulp mill and paper mills of Jämsänkoski and Kaipola on Central Lake Päijänne.A sulphite lye evaporation and combustion plant installed at the Jämsänkoski sulphite pulp mill in 1969 reduced the organic pollution of Central Päijänne. Lignin concentration decreased and the oxygen balance improved. The sulphite pulp mill was closed in 1981 and replaced by a thermo-mechanical pulp mill. Eutrophication is the main threat to water quality in Central Lake Päijänne at present.A significant improvement in the water quality, especially in oxygen balance, was achieved in the watercourse of Äänekoski and in Northern Päijänne after replacement of the old sulphite and sulphate pulp mills at Äänekoski by a large sulphate pulp mill with a biological purification plant employing the activated sludge method. The BOD₇-loading dropped from 46 to 3–4 t d^–1, but the nutrient loading has not decreased sufficiently, and the Äänekoski watercourse and Northern Päijänne are still eutrophic.     

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 g N⁻¹ m⁻³ ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mg l⁻¹, the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5 min, being independent of the concentrations, and the lower detection limit was 0.5 mg l⁻¹ BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD₅ method. Accordingly, a good correlation between the sensor and the conventional BOD₅ was obtained (r = 0.989). The system has to evolve the highly sensitive BOD determination.     

生物传感器快速测定BOD的研究

生化需氧量(biochemicaloxygendemand,BOD)是一种表征水体有机污染程度的综合指标,广泛应用于水体监测和废水处理厂的运行控制。由于BOD的标准测定方法需时5天,不能及时地反映水质状况和反馈处理信息,因此快速测定BOD的方法和仪器化研究近年来得到广泛的重视。利用生物传感器测定BOD是一种有效地快速测定废水中可生化降解有机物的方法。介绍生物传感器的工作原理及其生物敏感材料,讨论BOD传感器的性能参数以及BOD快速测定值(BODst)与标准BOD5值的一致性问题。对现阶段市场上常见的几种BOD快速测定仪进行简单的介绍,并对它们的性能进行比较 。     

Validation of the use of microbial consortium as standard seeding material in BOD determination

An attempt is made to validate the use of a microbial consortium in BOD analysis. A uniform dehydrated microbial consortium, `BODSEED', has been used as a seeding material in BOD analysis of synthetic and other industrial effluents. Statistical analysis of the obtained BOD values shows that conventional seeding material such as sewage can be replaced by `BODSEED'.