• Analysis of Feedstocks
    For AD and RNG Projects
    At Celignis Biomass Lab

Background to Anaerobic Digestion

For the past few decades, waste management has become one of the major challenges across the world. Waste disposal has been strictly regulated by governments in order to reduce soil, water, and air pollution caused as a result of landfilling and the direct disposal of wastes to water. Anaerobic digestion (AD), often referred to as Renewable Natural Gas (RNG) in North America, has been touted as a means of dealing with organic wastes while, at the same time, also addressing mankind’s energy needs. The process involves biogas production via the microbial degradation of organic compounds in the absence of oxygen. AD reduces the bulk of organic matter to be disposed, generates digestate rich in nutrients that can be of agricultural value, and produces biogas rich in methane which can be used as fuel directly or can be converted to compressed natural gas (CNG) and liquefied natural gas (LNG).

AD has a long history, with the first operational AD system constructed in India in 1897. Currently, there are a number of countries that each have thousands of biogas plants for domestic and commercial purposes. It is considered that the simplicity of AD, when compared against other biological and thermal technologies for the processing of organic wastes, coupled with its adaptability to a wide spectrum of feedstocks, that has led to its current scale of adoption. Crop and livestock farmers use AD plants for fuel production, manure management, and fertiliser production, whilst commercial AD plants can operate with a more diverse set of feedstocks, including municipal solid wastes (MSW) and industrial effluents, and their focus can often be on the reduction of chemical oxygen demand (COD) and effluent treatment as well as on energy generation.

Representation of the AD process

Advantages of Anaerobic Digestion

There are a number of advantages of using AD processes, however the two most important ones relate to the reduction/treatment of waste and to the valorisation of feedstocks.

Waste Treatment through AD/RNG

Both aerobic and anaerobic digestion degrade the organic matter present in the waste, but aerobic digestion is limited by oxygen supply and utilisation rate by microbes. The major advantages of the anaerobic process with regards to waste treatment relate to the fact that less sludge is retained after the process compared with aerobic digestion (so reducing residue treatment costs) and that AD allows for the possibility of higher organic loading rates. In most cases, AD also reduces the chemical oxygen demand (COD) of the waste stream significantly and it has also been noted that toxic compounds can be converted to their non-toxic forms by the process.

Disposing waste in open landfills can generate intense odours due to the formation of volatile malodorous compounds and can also contaminate the soil, water, and air with pathogens. AD can be used as a means for odour and pathogen reduction, depending on the operating conditions. It can convert the volatile compounds to methane and carbon dioxide, however poorly-managed AD facilities can result in the accumulation of volatiles, exacerbating odour problems. The efficacy of AD in pathogen removal depends on the process conditions and exposure time. Thermophilic digesters are best at removing pathogens from the waste, with mesophilic digestors taking longer to reduce pathogen levels. However, digesters operating at psychrophilic/ambient conditions may still have pathogens surviving at the end of the process.

There are a number of advantages of using AD processes, however the two most important ones relate to the reduction/treatment of waste and to the valorisation of feedstocks.

Valorisation of Feedstocks through Anaerobic Digestion

Biogas as an Energy Source

AD produces biogas as a result of its degradation of organic matter. Biogas composition is mainly methane (CH4, 50-70%) and carbon dioxide (CO2, 25-50%), with the remainder being nitrogen (N2), hydrogen (H2), oxygen (O2), and traces of hydrogen sulphide (H2S) and ammonia (NH3).

These two trace gases are responsible for the odour of the biogas and their concentration levels depend on nature of the feedstock and process conditions. They can be removed from the biogas using scrubbers with adsorbing or absorbing materials.

The energy content (lower heating value, LHV) of methane is 35.8 MJ/m3, and the LHV of biogas as a whole can range from 21.3 to 23.4 MJ/m3, depending on its methane content. Biogas is commonly used in combined heat and power (CHP) processes to produce heat and electricity. Depending on the digester type and the climate of the region in which the AD facility is located, some or all of the heat produced may be used within the plant to maintain the digester temperature at optimum levels. Biogas can also be upgraded to biomethane for the production of compressed natural gas (CNG) and liquefied natural gas (LNG), which have similar energy properties as fossil-fuel natural gas and can be used as transportation fuels. It is estimated that 1172 m3 of methane is equivalent to 1 tonne of oil equivalent.

Biochemical Steps in the AD Process

The four stages of anaerobic digestion
The quality of the biogas and digestate are highly dependent on the balance of the four stages in the AD process, namely: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The biochemical reactions that take place during these four stages are governed by the microbial community in the AD reactor, with the products and co-products formed at each stage having an effect on prior and subsequent stages. This means that a balanced microbial consortium of hydrolysing bacteria, acidogenic bacteria, acetogens and methanogens are required in the system. Similarly, the composition of the feedstock, or mixture of feedstocks, in the AD system, will impact upon the progression and efficiencies of each of the four stages.

In the first stage, hydrolysis, complex carbohydrates, proteins, and fats are hydrolysed to sugars, amino acids, and fatty acids, respectively. These monomers are then converted to volatile fatty acids (VFAs), alcohols, and gases in the second stage (acidogenesis). The concentration of VFAs has a significant effect on methanogenesis and is one of the key process indicators. In the third stage, acetogenesis, acids and alcohols are converted to acetic acid, hydrogen, carbon dioxide and other gases. In fourth and final stage, methanognesis, hydrogenotrophic methanogens utilise hydrogen and carbon dioxide or formate to produce methane whilst acetoclastic methanogens produce methane from acetate produced in the second and third stages.

In well-managed AD systems, all four stages are perfectly synchronised, for example with VFAs concentrations kept under control by active methanogenic bacteria. However, higher rates of acid formation compared to methane production will result in the accumulation of acids over time and lead to digester failure, a phenomenon known as “acid crash”. This situation generally occurs when there are high concentrations of easily-degradable sugars in the feedstock. Other problems may arise when there is a lot of nitrogen in the feedstock as this can result in high concentrations of ammonia, which is toxic for methanogens, being produced in the third stage (acetogenesis).

Feedstocks for Anaerobic Digestion

AD can be used to produce biogas and energy from a variety of organic feedstocks and also as a means of treatment and value-addition for various kinds of waste streams. The type of feedstock used is of great importance as it will influence the optimal AD process conditions as well as the quality of the biogas and digestate. AD facilities that rely on a small subset of agricultural residues and energy crops and on-site AD facilities that deal with the outputs of a particular industrial process tend to have relatively consistent supplies of reasonably homogeneous feedstocks. However, stand-alone AD plants that use feedstocks from a variety of sources tend to have inconsistent levels of supply as well as heterogeneous feedstock compositions that can cover wide ranges of levels in: complex carbohydrates, proteins, lipids, sugars, and acids.

Additionally, the storage of feedstock can have a big influence in its conversion in AD processes. Improperly-stored feedstock degrades in the storage area and generates acids and ammonia which can be toxic for AD above certain concentrations.

The properties of many feedstocks can be improved through pre-treatment prior to AD. This can involve: screening/sorting; size reduction by blending/milling; thermal treatment; and liquifaction, among other processes.

Analytical Requirements in Evaluating AD Feedstocks

Biomethane Potential

Feedstock analysis is crucial in order to properly evaluate feedstocks for use in AD processes. Of particular importance is the biomethane potential (BMP). This laboratory tests involves mixing the organic substrate with an anaerobic inoculum in a closed reactor that is incubated at a set temperature, with the contents mixed, for a set period of time. During this period the sample is digested and biogas is produced. The volume of biogas is monitored allowing for a cumulative plot of biogas production over time to be derived. This biogas can then be analysed for its composition, in particular the methane content, to allow the BMP to be determined. The BMP can be considered to be the experimental theoretical maximum amount of methane produced from a feedstock.

AD feedstock analysis at Celignis

Other Relevant Analyses

In addition to the BMP, it is important to understand the composition of the organic matter in the feedstock in order to determine optimal process conditions for its digestion. For example, differentiating between: structural sugars and soluble sugars, total proteins, and total fats will help to understand how the feedstock will progress across the 4 stages of AD. Nitrogen content is also particularly important, in order to balance the C/N ratio and avoid toxic levels of ammonia, and it is often the case that, based on the results of analysis, the co-digestion of two or more feedstocks may be necessary in order to balance the carbon-nitrogen ratio (C/N) in the AD system.

Other important parameters that should be tested for in a feedstock include toxic compounds such as acids, ammonia, and heavy metals. In the case of waste streams from industries, other specific compounds/intermediates released in trace quantities by the industrial process should also be determined as they can possibly accumulate in the digester with time and might inhibit the AD process.

It should also be considered that the composition of a given feedstock is not necessarily static, it can change according to variations in its supply and production. For example, the compositions of many samples can vary according to the time of year they are harvested/collected as well as depending on their length of time in storage. As a result, samples should be tested, for their composition and BMP, in a variety of conditions, where these may be considered to influence the outputs and course of digestion in AD processes.

How we Can Help with Your AD Analytical Requirements

Celignis is an analytical laboratory focused on the bioeconomy. We have developed a strong reputation for detailed and accurate analysis of lignocellulosic biomass and of the intermediates and outputs of biorefining processes, having analysed thousands of samples in this sector. We also have an extensive array of analysis packages relevant to the anaerobic digestion sector. These methods are based on the expertise of new CIO Lalitha Gotttumukkala who has worked for several years on determining properties relevant for AD and on optimising AD processes. With the user-friendly online Celignis Database you can monitor the results from our analyses even while the analysis is still ongoing. Our team of experts are also just a call/email away to discuss your results and provide advice.

Listed below are some of the analytical services we provide for evaluating AD feedstocks and processes:

Stoichiometric Methane Potential (SMP)

The maxmium theoretical methane potential possible from a given feedstock would be attained under conditions where all of the organic material of the sample was converted to biogas, with no residual digestate produced. We calculate this stoichiometric methane potential (SMP) using the Buswell Equation which uses the stoichiometric ratio of the products methane, carbon dioxide, ammmonia and hydrogen sulphide under the assumption that these are the only products from the complete breakdown of biomass of chemical composition of CHONS. The SMP is determined whenever analysis packages that involve the ultimate analysis (CHNS and ash) of a sample are undertaken.

Biomethane Potential (BMP)

Of course, the actual biomethane outputs of anaerobic digestion will always be less than the theoretical maximum calculated by the SMP as some of the biomass will be converted into microbial matter and also because it is unlikely that all of the organic matter (e.g. the lignin) will be fully converted. We therefore recommend that the biomethane potential (BMP) test is undertaken in order to get a better approximation of yields from a real AD system. We have FOUR Anaero BMP systems that allow us to digest your samples and determine the biogas yield over the course of the digestion which can last for 14, 21, 18, or 40 days. Our BMP analysis packages also determine the total solids (TS) and volatile solids (VS) contents of the sample and also involve periodic analyses of the composition of the biogas (for methane, carbon dioxide, hydrogen sulphide, oxygen, and ammonia) using the Biogas 5000 portable gas analyser.

AD Feedstock Analysis

Our analysis package P93 - Feedstock Chemical and Biological Analysis covers the important chemical and biological properties of the feedstock. Analytes that we determine include: Total Solids, Volatile Solids , pH, Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), Phosphorus, Potassium, Ammonia, Carbon, Hydrogen, Nitrogen and Sulphur.

Detailed Compositional Analysis of the Feedstock and Digestate

Using our expertise in the lignocellulosic analysis of biomass samples, we can offer an even more comprehensive profile of the chemical composition of both the feedstock and digestate. Under analysis package P10 we can determine the amount of extractives, lignin (Klason and acid soluble), ash, and lignocellulosic sugars (glucan, xylan, mannan, arabinan, galactan, and rhamnan) in the feedstock and digestate. Analysis package P12 can analyse the water extractives for soluble sugars, package P14 can determine the starch content of samples, and package P15 can quantify the amounts of five different uronic acids that are present. When such detailed analysis is undertaken on both the feedstock and the digestate (whether from the AD facility itself or from our BMP tests) it is possible to get a detailed picture regarding how each of the main biogenic polymers (cellulose, hemicellulose, lignin, and starch) are digested in the AD process. These observations can lead to process improvements targeted at maximizing biogas yields from components (cellulose, hemicellulose, and starch) that should be digested in efficiently-operating AD systems.

Additional Information on AD Feedstock Analyses

Feel free to get in touch with us if you have any questions about our analytical services or if you are looking to evaluate potential feedstocks for AD and RNG projects. Relevant members of the Celignis anaerobic digestion team will be happy to assist. Those team members with the most experience with undertaking these tests and interpreting the resulting data are listed below.

Lalitha Gottumukkala

Founder and Lead of Celignis AD, CIO of Celignis


Has a deep understanding of all biological and chemical aspects of anaerobic digestion. Has developed Celignis into a renowned provider of AD services to a global network of clients.

Kwame Donkor

AD Services Manager

BSc, MSc, Phd (yr 4)

His PhD focused on optimising AD conditions for Irish feedstocks such as grass. Kwame is now leading the Celignis AD team in the provision of analysis and bioprocess services.

Dan Hayes

Celignis CEO and Founder

PhD (Analytical Chemistry)

Dreamer and achiever. Took Celignis from a concept in a research project to being the bioeconomy's premier provider of analytical and bioprocessing expertise.

Other Celignis Tests and Services for Anaerobic Digestion

Global Recognition as AD/RNG Experts

Celignis provides valued services to over 1000 clients. We understand how the focus of AD projects can differ between countries and have advised a global network of clients on their RNG projects. We also have customs-exemptions for samples sent to us allowing us to quickly get to work no matter where our clients are based.

Further Info...

Biomethane Potential

The biomethane potential (BMP) can be considered to be the experimental theoretical maximum amount of methane produced from a feedstock. In our laboratory, we have six BMP systems, comprising 90 reactors, that allow us to digest your samples and determine the biogas yield over periods of between 14 and 40 days.

Further Info...

Continuous Digestions

To help you evaluate how well your anaerobic digestion feedstocks will behave in real-world conditions we can undertake continuous digestion experiments. These operate at scales up to 12 litres and typically run for 3 months. We target maximum achievable organic loading rate (OLR) and biomethane potential.

Further Info...

Toxicity Assays

The waste streams used in AD that arise from process industries may contain toxic or bacterial inhibitory compounds (e.g. antibiotics, polyelectrolytes, detergents). Our anaerobic toxicity assays can determine the presence of such toxicities and suggest the feeding limits for feedstocks.

Further Info...

Process Optimisations

There a many factors to consider when running an AD facility. We can design and experimentally-validate optimisations of these factors at the lab-scale prior to you implementing them at your AD facility. Such an approach allows for greater benefits and lower costs than optimising the process at the commercial scale.

Further Info...

Biological Consultations

We're experts in the biology of anaerobic digestion. We pour through operational data from biogas plants and identify correlations between process parameters and plant performance. This understanding on the specific biology of the digester allows for recommendations as to how peformance can be improved and made more stable.

Further Info...

Specific Microbial Activity

AD is a microbial process involving a sequence of stages (hydrolysis, acidogenesis, methanogenesis) to convert a complex feedstock to methane. We analyse samples collected from digesters and undertake tests to investigate how well they proceed with each of these stages of digestion.

Further Info...

Technoeconomic Analyses

Our TEA experts work with you to evaluate the economic prospects of your AD/RNG facility, considering various scale, technology, and feedstock options. We apply accurate costing models to determine CAPEX/OPEX of simulated and pilot scale processes which are then used to determine key economic indicators (e.g. IRR, NPV).

Further Info...

Digestate Analysis

Digestate is the residue after the anaerobic digestion process. It can potentially have value as a soil fertiliser. We offer a range of detailed analysis packages for digestate, allowing you to fully assess this resource and to determine the best use for it. Our team can also assist in evaluating digestate valorisation options.

Further Info...

Project Development

The criteria for the development of a successful AD project are numerous and vary according to region, technology, and feedstock. We have a deep understanding of these regional, technical, and biological differences and have advised a global network of clients on effectively developing their AD projects.

Further Info...

Process Parameters

Celignis can undertake a range of key analyses for KPIs and advanced process monitoring. These include volatile fatty acids (VFAs); Alkalinity ratio (FOS/TAC); and redox potential. It is particularly imporant that these are monitored when undergoing changes of feedstock type, organic loading rate and hydraulic retention times.

Further Info...

Nutrient Supplementations

Nutrients are essential for maintaining stable microbial populations and for efficient anaerobic digestion. We can suggest optimal values for the presence of major and minor elements in the digester as well as upper and lower threshold values. This allows us to formulate a bespoke cocktail of additives according to the requirements of the digester.

Further Info...

Publications on Anaerobic Digestion By The Celignis Team

Ravindran, R., Donkor, K., Gottumukkala, L., Menon, A., Guneratnam, A. J., McMahon, H., Koopmans, S., Sanders, J. P. M., Gaffey, J. (2022) Biogas, biomethane and digestate potential of by-products from green biorefinery systems, Clean Technologies 4(1): 35-50



Global warming and climate change are imminent threats to the future of humankind. A shift from the current reliance on fossil fuels to renewable energy is key to mitigating the impacts of climate change. Biological raw materials and residues can play a key role in this transition through technologies such as anaerobic digestion. However, biological raw materials must also meet other existing food, feed and material needs. Green biorefinery is an innovative concept in which green biomass, such as grass, is processed to obtain a variety of protein products, value-added co-products and renewable energy, helping to meet many needs from a single source. In this study, an analysis has been conducted to understand the renewable energy potential of green biorefinery by-products and residues, including grass whey, de-FOS whey and press cake. Using anaerobic digestion, the biogas and biomethane potential of these samples have been analyzed. An analysis of the fertiliser potential of the resulting digestate by-products has also been undertaken. All the feedstocks tested were found to be suitable for biogas production with grass whey, the most suitable candidate with a biogas and biomethane production yield of 895.8 and 544.6 L/kg VS, respectively, followed by de-FOS whey and press cake (597.4/520.3 L/kg VS and 510.7/300.3 L/kg VS, respectively). The results show considerable potential for utilizing biorefinery by-products as a source for renewable energy production, even after several value-added products have been co-produced.

Donkor, K. O., Gottumukkala, L. D., Lin, R., Murphy, J. D. (2022) A perspective on the combination of alkali pre-treatment with bioaugmentation to improve biogas production from lignocellulose biomass, Bioresource Technology 351



Anaerobic digestion (AD) is a bioprocess technology that integrates into circular economy systems, which produce renewable energy and biofertilizer whilst reducing greenhouse gas emissions. However, improvements in biogas production efficiency are needed in dealing with lignocellulosic biomass. The state-of-the-art of AD technology is discussed, with emphasis on feedstock digestibility and operational difficulty. Solutions to these challenges including for pre-treatment and bioaugmentation are reviewed. This article proposes an innovative integrated system combining alkali pre-treatment, temperature-phased AD and bioaugmentation techniques. The integrated system as modelled has a targeted potential to achieve a biodegradability index of 90% while increasing methane production by 47% compared to conventional AD. The methane productivity may also be improved by a target reduction in retention time from 30 to 20 days. This, if realized has the potential to lower energy production cost and the levelized cost of abatement to facilitate an increased resource of sustainable commercially viable biomethane.

Donkor, K. O., Gottumukkala, L. D., Diedericks, D., Gorgens, J. F. (2021) An advanced approach towards sustainable paper industries through simultaneous recovery of energy and trapped water from paper sludge, Journal of Environmental Chemical Engineering 9(4): 105471


This study considered the possibility of reducing the environmental footprint of paper and pulp industry by producing bioenergy from paper sludge by using process wastewater instead of fresh water, and reclaiming water trapped in paper sludge. Experimental studies are conducted with streams from three different pulp and paper mills (virgin pulp mill (VP), corrugated recycling mill (CR), tissue printed recycling mill (TPR)) for sequential bioethanol and biogas production with simultaneous reclamation of water from paper sludge (PS). Total energy yields of 9215, 6387, 5278 MJ/tonne dry PS for VP, CR and TPR, respectively, were obtained for ethanol-biogas production. Virgin pulp paper sludge gave the highest yield for ethanol and biogas in stand-alone processes (275.4 kg and 67.7 kg per ton dry PS respectively) and also highest energy conversion efficiency (55%) in sequential process compared with CR and TPR. Energy and environmental case study conducted on virgin pulp mill has proven the possibility of using paper sludge bioenergy to reduce energy demand by 10%, while reclaiming 82% of the water from the PS, reducing greenhouse gas emissions (GHG) by 3 times and producing solids suitable for land spreading.

Gottumukka L.D, Haigh K, Collard F.X, Van Rensburg E, Gorgens J (2016) Opportunities and prospects of biorefinery-based valorisation of pulp and paper sludge, Bioresource technology 215: 37-49


The paper and pulp industry is one of the major industries that generate large amount of solid waste with high moisture content. Numerous opportunities exist for valorisation of waste paper sludge, although this review focuses on primary sludge with high cellulose content. The most mature options for paper sludge valorisation are fermentation, anaerobic digestion and pyrolysis. In this review, biochemical and thermal processes are considered individually and also as integrated biorefinery. The objective of integrated biorefinery is to reduce or avoid paper sludge disposal by landfilling, water reclamation and value addition. Assessment of selected processes for biorefinery varies from a detailed analysis of a single process to high level optimisation and integration of the processes, which allow the initial assessment and comparison of technologies. This data can be used to provide key stakeholders with a roadmap of technologies that can generate economic benefits, and reduce carbon wastage and pollution load.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Pandey A (2014) Growth and butanol production by Clostridium sporogenes BE01 in rice straw hydrolysate: kinetics of inhibition by organic acids and the strategies for their removal, Biomass Conversion and Biorefinery 4(3): 277-283


Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resinsXAD 4, XAD 7, XAD 16, and an anion exchange resinSeralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.