Getting the best value from laboratory biodegradability testing by avoiding some common pitfalls.
The waste hierarchy, landfill pre-treatment requirements and landfill diversion targets are all driving reductions in the amount of waste going to landfill and promoting adoption of the best environmental outcome for waste. In-line with these drivers, the Environment Agency requires quantification of the reduction in the biodegradability of waste achieved by treatment to calculate landfill diversion of biodegradable waste. Good quality waste characterisation data are fundamental to achieving these aims.
Government initiatives to increase the re-use and recycling of wastes have provided a new lease of life to laboratory-based biodegradability tests. Biodegradability testing of waste materials can be an important way of assessing:
- the amount of biodegradable municipal waste (BMW) diverted from landfill by a given treatment;
- the stability of the waste material for re-use following treatment; and
- the potential biogas production potential of a waste material if it were utilised in an energy capture/ release process.
WRc have recently expanded their in-house testing facilities with a new suite of aerobic and anaerobic tests for assessing the wide range of waste outputs from mechanical biological treatment facilities, anaerobic digesters and composting processes.
Biodegradability testing is in principle not a complex procedure, in that it uses naturally occurring bacteria to degrade a given sample under aerobic or anaerobic test conditions. However, there are numerous pitfalls that the laboratory will need to overcome before it is possible to deliver meaningful and reproducible results.
The first hurdle to be faced by a waste producer who needs testing is the production of a suitable sample to send to the laboratory. Money spent on testing is completely wasted if the sample being tested is not representative of the source from which it was taken. Waste is commonly a heterogeneous material, that is also extremely variable over time and sample sizes need to be substantially scaled up over those traditionally used for example in soil testing.
In addition, plants can process thousands of tonnes of waste materials and the variability in inputs to the plant and the treatment process need to be properly assessed prior to developing a suitable sampling regime. The sample for laboratory analysis should be derived from a much larger composite sample collected over a suitable operating period at the plant. This means that considerable effort needs to be given to the sampling operation.
Shortcuts generate test data that is not consistent over time and reduces certainty in the efficacy of a process and confidence in the ability to meet targets. The composite sample should be reduced in size using an appropriate sub-sampling technique to produce a test sample for the laboratory. Where biological tests are required, the time between sampling and preparation for analysis should be kept to a minimum and under conditions that minimise biological changes in the sample, e.g. refrigerated storage in the dark.
If there is to be any significant period between collection and testing, the sample should be frozen until analysis can be started.
Representative sampling is key to the production of valid and consistent data. A robust sampling plan and good quality testing will help to ensure that reported variations in the biodegradability of the waste relates to waste material characteristics and plant performance, and that variability associated with sampling and testing are kept to a minimum.
Where testing is being undertaken for Landfill Allowance Trading Scheme (LATS) or Landfill Allowance Scheme (LAS) diversion monitoring purposes it is now necessary to separate out the biological component in the waste stream to produce a sample for testing whilst at the same time maintaining the integrity of the sample.
If you can imagine only taking a 5kg sample of municipal ‘black bin’ waste, it is easy to see that a leather shoe and a book of the yellow pages in your sample may lead to the organic fraction of the sample being less than representative of the biological components more commonly present in the municipal waste stream. To avoid bias at this stage, laboratory sample sizes are commonly measured in tens and, occasionally, hundreds of kilograms to ensure samples are representative.
Minimum sample size guidelines for processes treating municipal waste are provided in the Environment Agency Guidance 2009 ; these are based on empirical studies and practical experience. The determination of the percentage biodegradable waste (BW) content of waste is usually performed by manual sorting, a job that requires care and patience.
Commonly the waste sample may need to be separated into its component fractions, for example: paper, card, partially degradable fabrics, green waste and putrescibles and a fines fraction and the weight percent of the component fractions determined prior to reconstitution of a sample for testing. The separated biological (BW) fraction is then ready for sample preparation prior to testing.
“representative sampling is key to the production of valid and consistent data”
Most biodegradable waste fractions will often require sample preparation involving shredding, maceration, mixing and sub-sampling to provide a smaller representative sub-sample for analysis. Depending on available equipment for macerating the BMW, the samples may need to be bulk dried at a suitable temperature such as 70°C as some equipment does not effectively macerate wet material. Drying at this temperature also has the benefit of minimising further biological changes in the sample.
Preparation of the test sample commonly needs robust crushing and cutting equipment as some of the constituents in some streams can contain stones and small metal objects that can damage unsuitable equipment. The entire bulk sample is commonly shredded to <30 mm during the initial sample preparation; bulk dried, mixed and cone and quartered to give a suitable sub-sample for further particle size reduction by shredding/grinding to a particle size of <10 mm required for biodegradability tests.
Accurate determination of the dry matter (DM) and loss on ignition (LOI) contents are then required as a precursor to biodegradability testing as these data are used to determine the set-up conditions for the tests. Whilst DM and LOI content determination is a fairly routine analysis for both homogenous and heterogeneous materials for many laboratories, even when the wastes have been prepared to a small particle size of <10 mm it is necessary to ensure the test is undertaken on a representative sub-sample.
At least 100 g of material is needed, split into at least five replicates for waste samples. This compares with the 1 to 5 grammes commonly used for other more homogenous materials e.g. soil and plant samples.
We are finally at the point where a sample is ready for testing and a decision must now be made to select the most appropriate biodegradability test depending upon the overall objectives. The choice of either an aerobic or anaerobic biodegradability test could be important when considering questions like:
- the amount of BW diversion achieved by a given treatment for a variety of waste streams;
- the suitability of various wastes to a given treatment technology (e.g. composting, anaerobic digestion or thermal treatment);
- how quickly the results are required;
- if the testing is required for a trial or plant/waste stream characterisation or compliance monitoring; or finally for a designated re-use application.
The two most commonly adopted tests in the UK are:
- BMc (formerly BM100): an anaerobic biodegradation test used to determine the potential biogas production of the material under simulated landfill conditions
- DR4: a four day aerobic biodegradation test to determine the respiration activity or the potential oxygen consumed and carbon dioxide produced by the readily fermentable components of the BW fraction of the waste .
Although the principles behind biodegradability tests are relatively simple, the tests need to be set-up correctly using the right mix of sample, inoculum and nutrients to establish optimum conditions for growth of the desired micro-organisms (a form of selective microbial culturing). The use of replicates and appropriate quality control samples to check the performance and reproducibility of the run is also critical. WRc undertake all biodegradability testing in triplicate and run ‘real’ quality control samples to provide confidence in our data. WRc are in the final stages of UKAS accreditation of our laboratory testing procedures to show the importance we place on getting quality right for our clients.
BMc – Anaerobic Biodegradation Test
The BMc test reports biodegradability as litres of biogas produced per kg LOI and should be continued until biogas production ceases. This approach should optimise the calculated BMW diversion achieved by a biological treatment process. This test provides a measure of the potential amount of biogas (carbon dioxide and methane) that might be released by a waste material if it decomposed under the anaerobic methanogenic conditions that typify landfills for biodegradable waste. Under these conditions the decomposition of organic carbon produces biogas (carbon dioxide and methane).
Measuring the biogas production therefore directly measures the available organic carbon that is mineralised and thus the biodegradability of the test sample. The test is set up in a small vessel containing the test substrate, an aqueous mineral medium and an inoculum of digested sludge (that contains active methanogenic bacteria) taken from an active anaerobic digester. The test vessel is incubated at 35°C (the optimum temperature for growth of mesophilic methanogenic bacteria found in most landfills) and is monitored by collecting and recording the volume of biogas produced in a simple displacement tube.
The displacement liquid is a saturated saline solution, slightly acidified to help prevent absorption of carbon dioxide into the collecting liquid thereby ensuring all the biogas (carbon dioxide and methane) production is measured. This volume is then adjusted to standard temperature and pressure.
The test is incubated for an extended period until biogas production ceases, which may be up to (or occasionally greater than) 100 days depending on how well the test conditions have been optimised and how well a process has stabilised the test material, as more recalcitrant organics may take longer to degrade.
There are a number of commonly occurring problems with this type of test which can cause the test to stall or fail, some solutions to prevent such an eventuality are provided below:
• Some waste samples may be very water absorbent and swell up over the first few days leaving little, if any, free liquid inside the test vessel. If this occurs then the test samples should be topped up with additional deionised water until free liquid is observed.
• The contents of the reaction vessels need to be mixed regularly by swirling as some waste materials may ‘float’ on the surface of the main liquid. As this effectively separates the food source from the bugs, attention should be given to maintaining a well mixed test media.
• A common problem with highly biodegradable wastes (where an insufficient methanogenic bacteria population has been added via the seed) is that the test sample may become acidic due to the initial activity of fermentative (acetogenic) bacteria. These may produce more of the fermentation by-products than can be utilised by the methanogens and lead to acidification of the test solution which could then stall the test. The pH of the reaction mixture should be checked and if below 6.5, saturated sodium carbonate solution should be added to bring the pH back to 7.5. Opening of the vessels to the air should only be undertaken as a last resort to avoid stress to the methanogen population.
Overall the BMc test provides a valuable and reproducible measure of the potential biogas production of the sample and is the basis of the Environment Agency Guidance for LATS and LAS. Where an interim measure of biodegradability is required within the time-frame of the BMc test it is possible to use a short-term aerobic test such as the DR4. Also by running both tests simultaneously a plant specific correlation can be developed which, if satisfactory, can be used to produce surrogate BMc data.
DR4 – Aerobic Biodegradation Test
The four-day aerobic DR4 biodegradability test method provides a rapid assessment of the potential biodegradability of a test sample and like the BMc test is performed on the <10 mm particle size BW fraction. The method is based on the standard test method for measuring organic waste biodegradation (ASTM D5975-96). In this test a portion of the prepared sample is mixed with an equal amount (by dry weight) of mature green waste compost (as the inoculum), which provides an additional source of fermentative micro-organisms that are able to biodegrade the test material under suitable conditions.
The mixture is supplemented with nitrogen and phosphorus nutrients, the moisture content is adjusted, and then the mix is placed in an incubation vessel for four days at 35°C under aerobic conditions using forced aeration. Control vessels are also set up containing just the green waste seed material, an appropriate quality control substrate, and a blank vessel which is used to determine any carbon dioxide present in the air supply. During the four days of the test, the micro-organisms present in the sample and the green waste compost seed aerobically decompose some of the biodegradable organic carbon matter in the waste to CO2.
Consequently, the exhaust air leaving the reaction vessel is enriched with CO2. The amount of CO2 produced during the four-day test period is then measured by monitoring the output gases and the test results are converted to equivalent O2 consumption values by assuming there is a 1:1 molar ratio of CO2 to O2. The O2 consumed by the test is adjusted for the O2 consumption by the seed control and blank and the results are then reported as the DR4 value in units of gm O2/kg LOI; i.e. the amount of oxygen (in grammes) consumed over four days per kg of LOI of the test material.
There are a number of commonly occurring problems with this type of test which can cause the test to fail or produce lower gas yields, some solutions to prevent such an eventuality are provided below:
• The test samples are usually adjusted to 50% of the measured moisture content. However, some waste samples may be very water absorbent and dry out quickly especially when placed in incubators. If the samples are observed to be very dry after initial mixing, the water content should be adjusted accordingly.
• Equally, some samples may be too wet following mixing and form a thick paste. If this happens, the amount of water added should be reduced to ensure that the pore spaces are not closed off leading to a reduction in the rate at which air can be recirculated around the collection vessels and creation of anoxic/ anaerobic test conditions.
• The choice of DR4 apparatus chosen for the gas monitoring affects how well the gas consumption/ production can be monitored. Whatever set-up is chosen, for example, a sodium hydroxide (NaOH) trap system with titration or online gas analyzer system, it is important to ensure that all efforts are made to collect all available gas so as to minimise errors in the results.
• To prevent the samples drying out it is important that the flow rate of air through the vessels is the minimum sufficient to ensure carriage of emissions from the compost vessels, and that the incubator temperature is controlled at the recommended levels and that the temperature is maintained evenly throughout the incubator to ensure that test conditions are optimised for all the vessels.
In summary, to obtain best value from laboratory biodegradability testing, there are a number of key issues to consider:
1) Select a sampling approach that is appropriate to the objectives of the testing programme. These objectives will also guide the required analytical testing programme.
2) Representative sampling and sub-sampling are key to obtaining reliable and consistent results, whether they are to be used to assess plant performance in reducing waste biodegradability or to determine material characteristics to inform decisions about re-use.
3) Test the portion of the sample that is appropriate to the overall programme objectives. For assessing the biodegradability of municipal wastes, the non-biodegradable portion of the waste needs to be carefully segregated by sorting and quantified. This also means preparing the sample to an appropriate particle size. A minimum of <10 mm particle size is recommended in the Environment Agency Monitoring Guidance (2009). The use of small particle sizes ensures that there is a larger surface area available to enhance microbial interactions with the waste and thus achieve maximum biodegradation during the period of the test.
4) Laboratories should ensure that a representative sample is taken for the DM and LOI determinations and that replicate testing is employed. These values are used in calculating the sample amounts for the biodegradation test set-up and significant variations in the real sample DM and LOI values affect the reported values for DR4 or BMc tests.
5) Optimise test conditions for the biodegradability tests to ensure the reliability and reproducibility of test results.
6) Good laboratory practice, including use of suitable reference materials for Quality Assurance purposes, should be undertaken at all times and stringent quality checks followed prior to reporting of results.
Investment in good quality sampling and testing procedures will provide the waste producer with confidence that reported variability in the test data relates to the waste characteristics and not to artefacts of sampling and testing. This is especially the case for heterogeneous wastes where biological testing is required. Similar care should be taken in selection of an appropriate laboratory testing facility, evidence of a suitable track record in biodegradability tests is highly advantageous and where possible the laboratory should have accreditation through a recognised body for the waste matrix and tests required. These precautions will ensure that end-users have confidence in the test data, allowing them to make effective decisions about treatment plant performance and be sure that agreed targets can be met, or to base decisions about potential re-use or recycling options on sound science.
Note: The views expressed in this article are the authors’ own.
Jane Turrell, Principal Consultant and Nnenna Agbasiere, Environmental Consultant WRc
The WRc Group provides expert advice and solutions to meet the research, technical and business needs of the water, wastewater and environment industry.
For further information contact Jane Turrell at [email protected]
For more information on Lab Testing equipment please visit
Published: 10th Mar 2010 in AWE International