How to Assess Wastewater Biodegradability: Methods and Their Limits
The biodegradability of wastewater varies because its organic matter includes not only substances that micro-organisms readily decompose and use, but also substances that are hard to degrade or even inhibit microbial growth. The biodegradation behaviour of these organics and their relative abundance determine whether biological treatment — usually aerobic biological treatment — is feasible for a given wastewater, and how difficult it will be. Biodegradability also reflects how quickly the micro-organisms can use the pollutants: if the rate is so slow that treatment would take too long, the option is not practical in a real plant, and the wastewater is judged to be poorly biodegradable.
Aerobic Respiration Parameters
Aerobic degradation of organic pollutants changes water quality indicators such as COD and BOD, and at the same time consumes oxygen and generates carbon dioxide. Methods based on aerobic respiration exploit this: they measure changes in COD and BOD, or the consumption or production of oxygen and carbon dioxide and the rates at which these occur, to judge biodegradability. Three variants are in use: evaluation by water quality indicators, microbial respiration curves, and measurement of carbon dioxide production.
1. Evaluation by Water Quality Indicators
The BOD5/CODCr ratio is the classic and still the most widely used indicator. BOD is the oxygen consumed by aerobic micro-organisms as they metabolise organic pollutants, and BOD5 (five-day BOD) is taken to represent the biodegradable fraction. CODCr is the oxygen consumed by a chemical oxidant (potassium dichromate) in completely oxidising the organic matter, and is taken to represent the total organic content. The traditional view is that the B/C ratio expresses the proportion of biodegradable organic matter in the total, and can therefore be used to judge aerobic biodegradability: in general, the higher the BOD5/COD ratio, the more amenable the wastewater is to biological treatment.
Other organic indicators, notably total organic carbon (TOC) and total oxygen demand (TOD), can be measured instrumentally far faster and more reliably than COD and reflect organic content more accurately. As these methods have improved, BOD/TOD and BOD/TOC ratios have been adopted abroad as biodegradability indices, with their own sets of criteria. Whatever the denominator — COD, TOD or TOC — the principle is the same: the ratio of biodegradable to total organic matter is used to judge biodegradability.
The great strength of the approach is that BOD and COD are widely understood, the methods are mature and the equipment is simple. But it has clear weaknesses. BOD is an empirical parameter whose reproducibility and comparability depend on strictly identical test conditions: any deviation in dilution, analyst experience or seed material produces highly variable results, no standard seed exists against which to check the error introduced by the inoculum, and there is no way of knowing which measured value is closest to the true one. Reproducibility between laboratories for the same sample is in practice poor, largely because of differences in dilution water preparation and in local practice.
A further difficulty is that the BOD/COD ratio is not a true measure of the biodegradable fraction. Studies of industrial and municipal wastewaters have established relationships of the form COD = a + b·BOD, where a is the COD of the non-biodegradable fraction and b = CODB/BOD, CODB being the COD of the biodegradable fraction. It follows that BOD/COD is constant only when a is zero. Finally, some wastewater properties distort the ratio or even invert the conclusion: BOD cannot reflect inhibition by toxic substances, and where slowly degradable suspended or colloidal matter is present there is no good correlation between BOD and COD.
Three practical warnings follow from this.
(1) Suspended organic solids in some wastewaters are readily oxidised by dichromate and so appear in COD, but their physical form limits them in the BOD bottle, lowering BOD5 and hence the ratio. In reality such solids can be removed by bioflocculation and then hydrolysed by extracellular enzymes and oxidised within the cells, so a low BOD5/COD ratio does not necessarily mean poor biological treatability.
(2) COD includes oxygen consumed by inorganic reducing substances such as sulphides, sulphites, nitrites and ferrous iron, and BOD5 includes oxygen consumed by sulphides, sulphites and ferrous iron as well. Because the two methods differ, the final state and concentration of these substances differ too, so the oxygen they account for differs between the methods and directly affects both values and their ratio.
(3) Dichromate is a powerful oxidant under acid conditions and in most cases COD approximates the total organic content. Some compounds, however — pyridine, for example — are not oxidised by dichromate and so contribute nothing to COD, yet may be oxidised by micro-organisms and appear as BOD5, which strongly distorts the ratio.
In short, BOD5/COD is convenient but crude: it cannot equal the percentage of biodegradable organics, and an accurate conclusion should be supported by biological treatment model tests.
2. Microbial Respiration Curves
A microbial respiration curve plots oxygen uptake against time, and its shape depends mainly on the nature of the organic matter. Oxygen uptake rates are measured with a Warburg respirometer or an electrode dissolved-oxygen meter.
The endogenous respiration curve is the reference: once micro-organisms enter the endogenous phase their oxygen uptake rate is constant, uptake is proportional to time, and the plot is a straight line through the origin whose slope is the endogenous rate. Comparing the sample curve with this reference gives three diagnostic shapes. Curve a lies above the endogenous line, showing that the pollutants are biodegradable and that the uptake rate exceeds the endogenous rate; after a time curve a becomes almost parallel to the endogenous line, showing that substrate degradation is essentially complete and the biomass has entered endogenous respiration. Curve b coincides with the endogenous line, showing that the pollutants are not biodegradable but do not inhibit the biomass, which simply maintains endogenous respiration. Curve c lies below the endogenous line, showing that the pollutants are not biodegradable and are inhibitory or toxic; if the respiration curve meets the horizontal axis, respiration has stopped and the biomass is dead. Replacing time on the horizontal axis with substrate concentration converts the same experiment into the oxygen-uptake curve method — a different presentation of the same principle and procedure.
3. Measuring Carbon Dioxide Production
Degrading pollutants consumes oxygen and generates a corresponding quantity of carbon dioxide, so measuring CO2 production through the reaction indicates biodegradability. The most common approach is the Sturm test, run over 28 days, which compares actual with theoretical CO2 yield; the CO2/DOC ratio can also be used. Because the test needs special apparatus and is complex to run, it remains a laboratory method with no reported production-scale application.
Microbial Physiological Indicators
Once in contact with wastewater, micro-organisms use its organic matter as carbon and energy source. Physiological indicator methods observe changes in key biochemical indicators during that metabolism. The indicators most used are dehydrogenase activity and adenosine triphosphate (ATP).
Dehydrogenase activity. Oxidation of organic matter by micro-organisms is carried out by enzymes, among which dehydrogenases play a central role by catalysing the transfer of hydrogen from the substance being oxidised to another acceptor. Dehydrogenase is very sensitive to toxic substances, and its activity — the ability to activate hydrogen per unit time — falls when toxins are present. Dehydrogenase activity can therefore indicate the biomass's capacity to decompose pollutants: if activity rises in a culture grown on a given wastewater or organic pollutant, that substrate is degradable.
ATP. Oxidative degradation is essentially energy metabolism, and the biomass's capacity to produce energy directly reflects its activity. ATP is the energy-storage compound in microbial cells, so ATP level reflects the degree of microbial activity and serves as an indicator of degradative capacity: rising ATP activity in a culture grown on the substrate indicates that the substrate is degradable.
Other physiological indicators include standard plate counts, DNA measurement, INT reduction and luminescent-bacteria light intensity. Methods for dehydrogenase activity and ATP are reasonably mature, but the instrumentation and reagents required are demanding and the procedures complex, so these indicators are at present used mainly to judge the biodegradability and ecotoxicity of single organic pollutants.
Simulation Tests
Simulation tests judge feasibility by directly imitating the actual treatment process. Depending on how closely the simulation matches reality, they divide into culture-flask tests and model bioreactor tests.
Culture-flask (shaker) tests. A series of conical flasks is filled with culture medium using the pollutant or wastewater as carbon source, nutrients such as nitrogen and phosphorus are added, pH is adjusted, and one or more microbial strains — or acclimatised activated sludge — are inoculated. The flasks are shaken to simulate aerobic treatment, and over a period the physical appearance of the medium (concentration, colour, odour), the microbial population (species, biomass, phase) and indicators such as pH, COD or the concentration of the target pollutant are monitored continuously.
Model bioreactor tests. These run in a model bioreactor such as an aeration-tank model, reproducing the conditions of the real facility — MLSS, temperature, dissolved oxygen, F/M ratio — to predict removal performance and the influence of various factors.
Because simulation uses the same micro-organisms and wastewater as the real process and operates close to real conditions, it amounts to a pilot study of the treatment process. Factors that matter in practice all appear, the errors inherent in the other methods are avoided, and the conclusions are more reliable than those of culture-flask tests.
Integrated Modelling
Integrated modelling predicts the biodegradability of new organic compounds from correlations between molecular structure and the known biodegradability of large numbers of substances, using databases such as the EU EINECS inventory. The main models are BIODEG and PLS. The approach requires a very large database and is complex and expensive to run, so it is used mainly to predict the biodegradability of new compounds and their degradation pathways once released into the environment.
Other Approaches
Several further methods have been developed in recent years: using multistage filtration and ultrafiltration to obtain particle size distribution and COD distribution as predictors; combining oxygen consumption, end products of the biochemical reaction and biological activity values in a joint assessment; and using empirical flow charts to predict the biodegradability of a given organic pollutant.
In summary, each method has its strengths, and the right choice in practice depends on the nature of the wastewater and the available laboratory facilities.
