Appraising bacterial strains for rapid BOD sensing—an empirical test to identify bacterial strains capable of reliably predicting real effluent BODs

The measured response of rapid biochemical oxygen demand (BOD) biosensors is often not identical to those measured using the conventional 5-day BOD assay. This paper highlights the efficacy of using both glucose–glutamic acid (GGA) and Organisation for Economic Cooperation and Development (OECD) BOD standards as a rapid screen for microorganisms most likely to reliably predict real effluent BODs when used in rapid BOD devices. Using these two synthetic BOD standards, a microorganism was identified that produced comparable BOD response profiles for two assays, the MICREDOX® assay and the conventional 5-day BOD₅ test. A factorial experimental design systematically evaluated the impact of four factors (microbial strain, growth media composition, media strength, and microbial growth phase) on the BOD response profiles using GGA and OECD synthetic standard substrates. An outlier was identified that showed an improved correlation between the MICREDOX® BOD (BOD_sens) and BOD₅ assays for both the synthetic standards and for real wastewater samples. Microbial strain was the dominant factor influencing BOD_sens values, with Arthrobacter globiformis single cultures clearly demonstrating superior rapid BOD_sens response profiles for both synthetic and real waste samples. It was the only microorganism to approach the BOD₅ response for the OECD substrate (171 mg O₂?L^?1), and also reported BOD values for real waste samples that were comparable to those produced by the BOD₅ test, including discriminating between filtered and unfiltered samples.     

Effects of dilution on dissolved oxygen depletion and microbial populations in the biochemical oxygen demand determination

The biochemical oxygen demand (BOD) value is still a key parameter that can determine the level of organics, particularly the content of biodegradable organics in water. In this work, the effects of sample dilution, which should be done inevitably to get appropriate dissolved oxygen (DO) depletion, on the measurement of 5-day BOD (BOD₅), was investigated with and without seeding using natural and synthetic water. The dilution effects were also evaluated for water samples taken in different seasons such as summer and winter because water temperature can cause a change in the types of microbial species, thus leading to different oxygen depletion profiles during BOD testing. The predation phenomenon between microbial cells was found to be dependent on the inorganic nutrients and carbon sources, showing a change in cell populations according to cell size after 5-day incubation. The dilution of water samples for BOD determination was linked to changes in the environment for microbial growth such as nutrition. The predation phenomenon between microbial cells was more important with less dilution. BOD₅ increased with the specific amount of inorganic nutrient per microbial mass when the natural water was diluted. When seeding was done for synthetic water samples, the seed volume also affected BOD due to the rate of organic uptake by microbes. BOD₅ increased with the specific bacterial population per organic source supplied at the beginning of BOD measurement. For more accurate BOD measurements, specific guidelines on dilution should be established.     

Hydraulic loading,stability and water quality of Nakivubo wetland,Uganda

Nakivubo wetland, which has performed tertiary water treatment for Kampala city for the past 40 years, is ecologically stressed by agricultural and infrastructural developments. Field studies were carried out to assess the hydraulic loading, pollution profile, stability and water quality of this wetland. The upper and lower Nakivubo wetland receive 4.13?7.66 × 10⁴ and 3.50?10.32 × 10⁴m³/day of water respectively, of which 48.3–57.9% of total hydraulic loading to the upper wetland was carried by sampling station S1. The influent water to the upper wetland had a total BOD₅ and NH₄-N loading ranging from 2.6?4.4 × 10³kg BOD/day and 0.79?1.68 × 10³kg NH₄-N/day respectively. The National Water and Sewerage Corporation's effluent constituted a large proportion of BOD and NH₄-N loading into Nakivubo wetland. Zinc, copper and chromium were detected in trace amounts at most sampling stations. However, lead was occasionally detected at Kibira channel (station S5) at a concentration of 0.4mg/l, which is higher than the permitted Ugandan discharge limit of 0.1mg/l (NEMA 1999). The wetland showed a very high removal efficiency for BOD, ranging from 77.4%–86.3%, compared to ammonium-N which ranged from ?66.1% to 33.1% indicating limitations with the nitrification process. A low self-purification for zinc, copper and chromium was also observed in the upper Nakivubo wetland, possibly due to poor plant-wastewater interaction resulting from wetland drainage. In the lower Nakivubo wetland conductivity and dissolved oxygen were generally higher in papyrus- than in Miscanthidium-vegetated zones. However, the BOD and ammonium-N loadings did not vary significantly (P = 0.217 and P = 0.359 respectively) between the two vegetated zones.     

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100?mg for 2?L culture, twice that of Myrothecium verrucaria. The k _cat/K _m ratio for conjugated bilirubin (1,513?mM^?1?s^?1) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980?mM^?1?s^?1), with the lowest K _m measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300?min at 37?°C, high stability at pH?7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.     

Determination of chemical biodegradation by direct and indirect methods

Summary Degradation of 10 organic chemicals by pre-acclimated microorganisms in BOD dilution water was determined directly by UV spectrophotometry and indirectly by a modified BOD method. Residual chemical concentrations were periodically measured and pseudo-first-order biodegradation rate constants (k ₁) were calculated. Thek ₁ spectrophotometry values ranged from 0.006/h to 0.077/h andk ₁-BOD values from 0.002/h to 0.043/h for 1-methylnaphthalene and indole, respectively. The ratios ofk spectrophotometry to k₁-BOD were between 1.5 for salicylic acid and 3.0 for 1-methylnaphthalene with a mean of 2.7. A significant (=0.001) linear correlation (r ²=0.854,F=46.630) existed between the two sets of rate constants. Results from this study suggest that the modified BOD method may be used to estimate chemical biodegradation rates in synthetic media.     

Biochemical mediator demand – a novel rapid alternative for measuring biochemical oxygen demand

The biochemical oxygen demand (BOD) test (BOD₅) is a crucial environmental index for monitoring organic pollutants in waste water but is limited by the 5-day requirement for completing the test. We have optimised a rapid microbial technique for measuring the BOD of a standard BOD₅ substrate (150 mg glucose/l, 150 mg glutamic acid/l) by quantifying an equivalent biochemical mediator demand in the absence of oxygen. Elevated concentrations of Escherichia coli were incubated with an excess of redox mediator, potassium hexacyanoferrate(III), and a known substrate for 1 h at 37 °C without oxygen. The addition of substrate increased the respiratory activity of the microorganisms and the accumulation of reduced mediator; the mediator was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. Catabolic conversion efficiencies exceeding 75% were observed for the oxidation of the standard substrate. The inclusion of a mediator allowed a higher co-substrate concentration compared to oxygen and substantially reduced the incubation time from 5 days to 1 h. The technique replicates the traditional BOD₅ method, except that a mediator is substituted for oxygen, and we aim to apply the principle to measure the BOD of real waste streams in future work. Received: 2 August 1999 / Received revision: 6 December 1999 / Accepted: 12 December 1999     

A fast estimation of biochemical oxygen demand using microbial sensors

Summary Microbial amperometric sensors for biochemical oxygen demand (BOD) determination using Bacillus subtilis or Trichosporon cutaneum cells immobilized in polyvinylalcohol have been developed. These sensors allow BOD measurements with very short response times (15–30s), a level of precision of ±5% and an operation stability of 30 days. A linear range was obtained for a B. subtilis-based sensor up to 20 mg/l BOD and for a T. cutaneum-based sensor up to 100 mg/l BOD using a glucose/glutamic acid standard.     

Amperometric estimation of BOD by using living immobilized yeasts

Summary A microbial electrode consisting of immobilized living whole cells of yeasts, porous membrane and an oxygen electrode was prepared for continuous estimation of biochemical oxygen demand (BOD). Immobilized Trichosporon cutaneum was employed for the microbial electrode sensor for BOD. When a sample solution containing the equivalent amount of glucose and glutamic acid was injected into the sensor system, the current of the electrode decreased markedly with time until steady state was reached. The response time was within 18 min. A linear relationship was observed between the current decrease and the concentration below 41 mg l ^– of glucose and 41 mg l ^– glutamic acid (5-day BOD 60 mg l ^–). The current decrease was reproducible within ± 6% of the relative error when a sample solution containing 27 mg l ^– of glucose and 27 mg l ^– of glutamic acid (5-day BOD 40 mg l ^–) was employed. The microbial electrode sensor was applied to untreated waste waters from a fermentation factory. Good comparative results were obtained between BOD estimated by the microbial electrode and that determined by the conventional 5-day method (regression coefficient was 1.2). Furthermore, the effect of various compounds on BOD estimation was also examined. The current output of the microbial electrode sensor was almost constant for 17 d and 400 tests.     

Investigating CH4 and N2O emissions from eco-engineering wastewater treatment processes using constructed wetland microcosms

Methane (CH₄) and nitrous oxide (N₂O) are important greenhouse gases, because of their contribution to the global greenhouse effect. The present study assessed emissions of N₂O and CH₄ from constructed wetland microcosms, planted with Phragmites australis and Zizania latifolia, when treating wastewater under different biological oxygen demand (BOD) concentration conditions. The removal rate was 95% for BOD and more than 80% for COD in all three pollutant concentrations, both plants’ removal rates of pollutants were at almost the same level, and both were found to resist BOD concentrations as high as 200 mg L⁻¹. When BOD concentrations fell below 200 mg L⁻¹, the soil plant units reached an average of 80–92% T-N and T-P removal rates; however, as the concentrations increased to 200 mg mg L⁻¹ or when during the initial phases of winter, the removal rates for T-N and T-P decreased to less than 70%. With NH₃-N removal, the influences of BOD concentrations and air temperature were more obvious. When BOD concentrations increased to 100 mg L⁻¹ after October, an obvious decrease in NH₃-N removal was detected; almost no nitrification occurred beginning in December at BOD concentrations of 200 mg mg L⁻¹. N₂O and CH₄ emissions showed obvious seasonal changes; higher emissions were observed with higher BOD concentrations, especially among Z. latifolia units. The enumeration of methane-oxidizing bacteria and methane-producing bacteria was also conducted to investigate their roles in impacting methane emissions and their relationships with plant species. The pollutant purification potentials of P. australis and Z. latifolia plant units during wastewater treatment of different pollutant concentrations occurred at almost the same levels. The nutrient outflow and methane flux were consistently higher with Z. latifolia units and higher concentrations of BOD. The more reductive status and higher biomass of methanogens may be the reason for the lower nitrification and higher CH₄ emissions observed with Z. latifolia units and higher concentration systems. The Z. latifolia root system is shallow, and the activity of methanotrophs is primarily confined to the upper portion of the soil. However, the root system of P. australis is deeper and can oxidize methane to a greater depth. This latter structure is more favorable as it is better for reducing methane emissions from P. australis soil plant systems.     

Effect of operational and design parameters on removal efficiency of pilot-scale FWS constructed wetlands and comparison with HSF systems

In order to investigate the effect of temperature, hydraulic residence time (HRT), vegetation type, substrate material and wetland shape on the performance of free-water surface (FWS) constructed wetlands treating wastewater, 5 pilot-scale units were constructed and operated continuously from December 2004 until March 2007 in parallel experiments. Four of the units (A, B, C, D) were rectangular in plan view with dimensions 3.40 m in length and 0.85 m in width, and contained substrate material at a thickness of 0.45 m. The fifth unit (E) had a trapezoidal plan view shape, with a width at the inlet of 1.15 m and at the outlet of 0.55 m, while the length and the thickness of the substrate were the same as in the other four. All units operated at a water depth of 0.10 m. Units B–E contained clay substrate and unit A contained sand. The four units with clay were planted as follows: two with cattails (B and E), one with common reeds (C), and one with giant reeds (D). Unit A, containing sand, was planted with cattails. Planting and substrate material combinations were appropriate for comparison of the effect of vegetation and material type on the function of the system. Synthetic wastewater was introduced in the units. During the operation period four HRTs (i.e., 6 days, 8 days, 14 days and 20 days) were used, while wastewater temperatures varied from about 0.0 °C to 29.1 °C. The removal performance of the five constructed wetland units was good, since it reached on the average 77.5%, 67.9%, 60.4%, 53.9%, 56.0% and 51.7% for BOD, COD, TKN, ammonia (NH₄-N), ortho-phosphate (PO₄-P) and total phosphorus (TP), respectively. BOD and phosphorus removal efficiencies showed dependence on temperature in most units. The 14-day HRT was found adequate for acceptable removal of organic matter, nitrogen and phosphorus for most temperatures. A 20-day HRT is recommended for acceptable removal of BOD and PO₄-P in the cold season. The unit with the trapezoidal plan view shape showed the best performance, with mean removals of 80.1%, 73.5%, 70.4%, 68.6%, 64.7% and 63.5% for BOD, COD, TKN, NH₄-N, PO₄-P and TP, respectively. The cattail was found statistically more efficient than the other two plants in COD and PO₄-P removal. The unit that contained the clay substrate was found statistically more efficient in phosphorus removal than the unit containing sand. HSF CW units were found more efficient than FWS units in removal of most pollutant.     

Advanced treatment of high-strength wastewater by a submerged bio-film process

A submerged bio-film process was studied as a tertiary treatment to decrease the BOD of secondary effluent from a combined chemical-biological treatment of high-strength wastewater to below the permitted limit (30 mg/l) for waste facilities in Japan. The operational conditions to keep the final effluent below 30 mg/l of BOD were as follows: the influent BOD to the bio-film tank should be under 300 mg/l and a BOD-surface loading should be below 2 g/m²/d. The influent pH of 9.0–12.5 did not affect the treatment efficiency unfavorably. Acclimation is the compensating factor for variations of pH in this process. Removal rates of suspended solids were not influenced by BOD-surface loading. The calculated BOD removal coefficient was 2.57 × 10⁻⁵ when the influent BOD was below 300 mg/l.     

Some aspects of the metabolism of a partially colloidal substrate

The biochemical oxygen demand (BOD) of a complex, partially colloidal substrate is divided into oxidation (f_o) and synthesis (f_s) fractions. The substrate being investigated is a suspension containing 10 mg./l. of dry skim milk. The oxidized and synthesized fractions of the BOD are shown to be 38.4% and 61.6%, respectively. The division of BOD is related to the time at which maximum cell growth appears. The time required to obtain maximum cell production is shown to be at least 10 hr. The relationship between the time to maximum cell production and the apparent initial activity of the seed organisms is discussed.     

Lability of organic carbon in lakes of different trophic status

1. We used first‐order kinetic parameters of biological oxygen demand (BOD), the constant of aerobic decomposition (k) and the asymptotic value of BOD (BOD_ult), to characterise the lability of organic carbon pools in six lakes of different trophic state: L. Naroch, L. Miastro and L. Batorino (Belarus), L. Kinneret (Israel), L. Ladoga (Russia) and L. Mendota (U.S.A.). The relative contributions of labile and refractory organic carbon fractions to the pool of total organic carbon (TOC) in these lakes were quantified. We also determined the amounts of labile organic carbon within the dissolved and particulate TOC pools in the three Belarus lakes. 2. Mean annual chlorophyll concentrations (used as a proxy for lake trophic state) ranged from 2.3 to 50.6 μg L⁻¹, labile organic carbon (OC_L = 0.3BOD_ult) from 0.75 to 2.95 mg C L⁻¹ and k from 0.044 to 0.14 day⁻¹. 3. Our data showed that there were greater concentrations of OC_L but lower k values in more productive lakes. 4. In all cases, the DOC fraction dominated the TOC pool. OC_L was a minor component of the TOC pool averaging about 20%, irrespective of lake trophic state. 5. In all the lakes, most (c. 85%) of the DOC pool was refractory, corresponding with published data based on measurements of bacterial production and DOC depletion. In contrast, a larger fraction (27–55%) of the particulate organic carbon (POC) pool was labile. The relative amount of POC in the TOC pool tended to increase with increasing lake productivity. 6. Long‐term BOD incubations can be valuable in quantifying the rates of breakdown of the combined particulate and dissolved organic carbon pools and in characterising the relative proportions of the labile and recalcitrant fractions of these pools. If verified from a larger number of lakes our results could have important general implications.     

Measuring aquatic organic pollution by the Permanganate Value method

Permanganate Value methods provide a convenient procedure for measuring aquatic organic pollutants, and are compared with the Biochemical Oxygen Demand procedure

The educational convenience, safety, and results from Permanganate Value (PV) methods for measuring aquatic organic pollution were compared with those of the Biochemical Oxygen Demand (BOD) procedure, which is the standard in the water industry. Twenty river water and 20 sewage effluent samples were used in the investigation. Statistical relationships between results were calculated. It was found with the river water, whose BOD levels varied from 2–20 mg O₂ dm^?3, that there was no significant correlation between BOD and PV results. However, with sewage effluent, whose BOD levels varied from 7–330 mg O₂ dm^?3, the correlation coefficient between methods was 0.95 (p <0.001). With the PV method, reducing the incubation period to 20 min at 60°C gave simitar results to those from the usual period of 4 hours at 27°C. The shortened procedure should albw organic pollutants to be determined within the timespan of an educational practical session. Further educational investigations are discussed.     

Optimization of biodrying pretreatment of municipal solid waste and microbial fuel cell treatment of leachate

The biodrying pretreatment of municipal solid waste (MSW) and the treatment of leachate were investigated. The biological oxygen demand (BOD) and NH₄ ⁺-N concentration of leachate from MSW biodrying pretreatment were measured, and the optimal conditions for MSW biodrying pretreatment and microbial fuel cell (MFC) performance were established. The results show that the optimal temperature and time for biodrying pretreatment of MSW were 40°C and 6 day, resulting in 30% weight loss of MSW, 20,800 mg/L leachate BOD, and 1,410 mg/L leachate NH₄ ⁺-N. Effects of leachate properties on MFC performance were then studied. The optimal conditions for electricity generation of the MFC were neutral pH, 5,093 mg/L leachate BOD, and 341 mg/L leachate NH₄ ⁺-N. The stable voltage of MFC generated using diluted leachate was 0.32 V, and the removal efficiencies of BOD and NH₄ ⁺-N by the MFC were 86.0 and 88.8% after 7 day of treatment, respectively. These findings provide guidelines for the pretreatment of MSW and the treatment of leachate, and for further research and actual engineering application.     

A rational approach for predicting the dissolved oxygen profile in receiving waters

A practical method for predicting the dissolved oxygen (DO) profile in a stream receiving biodegradable waste is presented. In this method the BOD (accumulated O₂ uptake) curve is obtained using an open jug technique. The accumulated O₂ uptake curve thus developed is employed in numerical integration with the physical reaeration data for the receiving stream to predict the DO profile in the stream. In the present study, the method was examined using 10-liter open jar reactors to obtain the O₂ uptake curves, and the receiving stream was a 670-liter simulated stream apparatus which has been employed in previous studies on stream reaeration. The method was found to provide a fairly good prediction of the actual dissolved oxygen profile observed in the receiving stream. The effect of the reaeration constant, K₂ )agitation effect(, on the kinetics of O₂ uptake was also investigated and it was found that increased agitation (higher K₂ value) caused some increase in the accumulated oxygen uptake (BOD) curve with most of the increase coming after the so-called “plateau” area in the O₂ uptake curve, i.e., in the general case, after the low point along the DO sag curve.     

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.     

Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells

The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H₂O₂, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzo­thiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O₂ reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O₂ and/or H₂O₂ as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm^?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass.     

Development and Evaluation of an Online CO2 Evolution Test and a Multicomponent Biodegradation Test System

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO₂) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO₂ method with existing CO₂ evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO₂ evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO₂ and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.