Bioresource Technology xxx (2016) xxx–xxx

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Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Short Communication

Early assessment of a rapid alternative method for the estimationof the biomethane potential of sewage sludge

http://dx.doi.org/10.1016/j.biortech.2016.01.1390960-8524/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +33 (0)4 67 75 29 90.E-mail address: solenn.bellaton@envolure.com (S. Bellaton).

Please cite this article in press as: Bellaton, S., et al. Early assessment of a rapid alternative method for the estimation of the biomethane potentialage sludge. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.139

Solenn Bellaton a,⇑, Sabrina Guérin b, Nathalie Pautremat a,c, Jean Bernier b, Mathieu Muller a,Stéphane Motellet d, Sam Azimi b, André Pauss d, Vincent Rocher b

aAMS Envolure, 1682 rue de la Valsière, 34184 Montpellier Cedex 4, Franceb SIAAP, Direction du Développement et de la Prospective, 82 Avenue Kléber, 92700 Colombes, Francec SCANAE, 1682 rue de la Valsière, 34790 Grabels, FrancedCentre de Recherche Royallieu, Université Technologique de Compiègne, BP 20529, rue Personne de Roberval, 60205 Compiègne Cedex, France

h i g h l i g h t s

� Fast fluorescence-based method to investigate biodegradability of sewage sludge.� Proportionality between fluorescence and metabolic activity of anaerobic sludge.� Assessment of nineteen municipal sewage sludge by this method and by AMPTS.� Similar results between AMPTS and rapid alternative method for BMP measurement.

a r t i c l e i n f o

Article history:Received 20 November 2015Received in revised form 18 January 2016Accepted 19 January 2016Available online xxxx

Keywords:Anaerobic digestionBiodegradabilityBiomethane productionFluorescent redox indicatorAutomatic Methane Potential Test System

a b s t r a c t

This short communication briefly presents a rapid method using a fluorescent redox indicator, similar toresazurin, in order to estimate the biodegradability of sewage sludge during anaerobic digestion (AD).The biodegradability and by extension the Biochemical Methane Potential (BMP) of nineteen municipalsludge samples (primary, biological and tertiary) were investigated and estimated in only 48 h. Resultsshowed the relevance to follow the metabolic activity of anaerobic sludge by the kinetic of probe reduc-tion. The extended lag phase of inoculum indicated an impact of pre-treatments on enzyme activity. Thecomparison with Automatic Methane Potential Test System II (AMPTS) confirmed the estimated values ofBMP according to an uncertainty limit of 25%. These first results highlight the interest of this rapid assayas a preliminary tool of the biodegradability of sewage sludge in anaerobic digestion.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

The global population growth increases the demand for waterresources. Consequently, many research programs on tomorrow’swastewater treatment plants are conducted on each process stepin order to answer water and energy challenges (Ruffino et al.,2015). For that purpose, the MOCOPEE (MOdeling, Control andOptimization of wastewater treatment ProcEssEs) project focuseson applied metrology, modeling and on remote monitoring ofwastewater treatment process (www.mocopee.com).

Anaerobic digestion (AD) is a natural biological process wheremicro-organisms break down organic matter in the absence ofoxygen. Since few decades, AD is recognized as a renewable energy

having a huge potential. The anaerobic digestion process forwastewater treatment plants (WWTPs) is an economical processand is considered as being a major and essential part of WWTPs(Appels et al., 2008). Indeed, AD plays an important role thanksto its abilities to produce a net energy gain in the form of methanegas, to reduce the volume, the pathogens content and the odour ofsewage sludge (Silvestre et al., 2015).

The BMP assay is an important tool and the most commonlyused for the assessment of the anaerobic biodegradability of sew-age sludge produced during wastewater treatment (Aquino et al.,2008; Mottet et al., 2010). The BMP assay requires 20–60 days tomeasure the BMP of samples. Nevertheless, the required time is amajor inconvenience in answering operational requests in WWTPs.To address this lack of a rapid and pertinent tool, many develop-ments of alternatives approaches were made: modeling with theIWA ADM1 model (Batstone et al., 2002), or methods based only

of sew-

2 S. Bellaton et al. / Bioresource Technology xxx (2016) xxx–xxx

on organic matter characterization using spectroscopy (Jimenezet al., 2014; Lesteur et al., 2010). Recently, new tools, based on flu-orescence measurement, have been developed in order to answeroperational requests such as toxicity (Chen et al., 2015) or forhigh-throughput characterization of more than 32 samples simul-taneously in 48 h, for digester operation, the Envital� kit. More pre-cisely, these technologies use a probe where the kinetics of redoxindicator reduction is directly correlated with the activity of anaer-obic sludge (Chen et al., 2015). Furthermore, the few BMP data ofsewage sludge based on the existing literature are variable(Carrère et al., 2010; Jensen et al., 2014). According to Wanget al. (2015a), it is very difficult to compare these varying resultsbecause there are many differences in instrumentation, protocols,and different experimental conditions. The MOCOPEE researchprogram has worked on the determination of the BMP of sewagesludge with the Automatic Methane Potential Test System II, since2013. The aim is to provide a sewage sludge data base and to devel-oped mathematic models to predict methane production.

The objective of this short communication is to succinctly pre-sent a rapid assay based on fluorescence, the Envital

�kit, in order

to estimate anaerobic biodegradability of sewage sludge and tocompare it to AMPTS, in the context of MOCOPEE researchprogram.

2. Methods

2.1. Substrates and inoculum: characteristics and preparation

2.1.1. CharacteristicsThe inoculum, an anaerobic sludge, was systematically col-

lected from the same secondary digester on a WWTP next to Paris(interdepartmental association for sewage disposal of Paris conur-bation, SIAAP) but not necessarily the same day for both technolo-gies. Indeed, the Envital� experiments were carried out in parallelof the AMPTS analysis but also at different times. Thus, differentinocula were used between the two technologies. In the sameway, samples were collected from the WWTPs and at differentlocations in the process: primary settler (primary sludge), biologi-cal tank or biofilter (biological sludge), tertiary settler with chem-icals (tertiary sludge). Combinations of primary and biologicalsludge were sampled (mixed sludge). Physical and chemical prop-erties of the samples and inoculum, Total Solid (TS), Volatile Solid(VS), and Chemical Oxygen Demand (COD), have been measuredaccording to standardized methods (APHA, 2005) (Table1).

2.1.2. PreparationFor the AMPTS analysis, fresh samples and fresh inoculum have

been added in glass bottles of 500 mL each, following the [I]/[S]ratio of 3 based on VS content. This ratio is widely quoted in theliterature (Jackowiak et al., 2011) and advised by the manufacturer.40 mL of each sample was frozen in order to create a sample collec-tion stored at �20 �C for the comparison with Envital� assay inmicroplate. The freezing allowed to stabilize and conserve the

Table 1Principals characteristics of sewage sludge and inoculum.

Sewage sludge TS (g/L)(mean ± sd*)

VS (%TS)(mean ± sd*)

COD (g/gVS)(mean ± sd*)

Primary sludge 30.7 ± 11.6 79.3 ± 3.1 1.8 ± 0.1Biological sludge (tank) 34.2 ± 2.2 81.1 ± 4.3 1.5 ± 0.4Biological sludge (biofilter) 42.7 ± 16.9 72.3 ± 10.6 1.5 ± 0.4Mixed sludge 37.0 ± 0.4 77.4 ± 4.7 1.7 ± 0.2Tertiary sludge 29.7 ± 12.1 79.3 ± 3.1 1.8 ± 0.3Inoculum (anaerobic sludge) 22.7 ± 2.8 62.2 ± 6.6 1.7 ± 0.1

* sd: standard deviation.

Please cite this article in press as: Bellaton, S., et al. Early assessment of a rapidage sludge. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.201

organic matter on several weeks. Just before the Envital� analysis,40 mL of samples were gently thawed at room temperature and,then suspended and diluted in distilled water. For the Envital�

analysis, the anaerobic sludge, used as inoculum, was pre-incubated at 35 �C after collection from the mesophilic digesterfor 24 h. The pre-incubation of the anaerobic sludge allowed toreduce its organic content before using it in the BMP tests. Afterincubation, the inoculum was prepared by filtration with a specificfilter supplied in the kit in order to remove organic matter insuspension.

2.2. AMPTS

In a first step, the study of characterization of sludge sampleswas carried out using AMPTS II since 2013 (Bioprocess Control,Lund, Sweden). This technology allows automatic and real-timemeasurements of the biogas production during 20–40 days at35 �C ± 0, 2. Each reactor is mixed by a stirring rod in a circularmanner by a motor located on the top of the reactor (rotationspeed = 80 rpm � agitation frequency: 60 s ON/60 s OFF). The bio-gas produced was further led to a scrubbing reactor by flexible tub-ing. The acid gases (CO2, H2S) were trapped by an alkali solution(NaOH, 4 M) and the remaining biogas (methane) passed into themeasuring cell, containing a gas counter based on the principleof liquid displacement (Bassard et al., 2014). Gas bubbles generateimpulsions. These impulsions were recorded and translated in NmlCH4/gVS by AMPTS� v5 software. The temperature and pressuresensors are incorporated for a correction online of results. Blankscontaining only inoculum and without substrate were used to cor-rect for background methane potential.

2.3. Envital� kit

The Envital� kit, developed by Envolure, was used on all sam-ples following the recommended procedure with a given specificratio for: fluorescent probe, buffer solution, samples or standardsand inoculum in separate wells of 96-well microplates. In the sameway, blanks were used to correct for background methane poten-tial. To ensure anaerobic conditions, the wells were sealed imme-diately with a specific reagent and a microplate lid supplied inthe kit. Excepted for inoculum, all the reagents, standards andmaterials are provided in the kit. Analysis was carried out at35 �C during 48 h. Fluorescence was recorded every 30 min by afluorescence microplate reader (FLx800, BioTek�, USA) at kEx.= 540 nm and kEm. = 600 nm with a gain of 35. There was a shak-ing before each reading. The raw fluorescence data were translatedin Nml CH4/g VS thanks to the range of standard solutions (devel-oped by AMS Envolure, France) according to a linear regressionmodel (Fig. 1).

3. Results and discussion

3.1. Fluorescence and anaerobic biodegradability with Envital�

technology

WWTPs generate different sludge as a by-product of differentprocesses (primary, biological and tertiary treatment) used in thesewage treatment. Four sludge produced at different steps in theprocess were chosen in order to assess the ability of fluorescencetechnology to distinguish the different sewage sludge. Fig. 2(a)shows the cumulative fluorescence intensity resulting from themetabolic activity of anaerobic sludge. The control (blanks) repre-sented the endogenous metabolic activity without addition oforganic matter. The cumulative fluorescence intensity curves cor-responding to sludge samples were much higher than the control.

alternative method for the estimation of the biomethane potential of sew-6.01.139

Fig. 1. Scheme of an example of Envital� assay in microplate based on fluorescence measurement.

S. Bellaton et al. / Bioresource Technology xxx (2016) xxx–xxx 3

This fact confirmed the correlation between the kinetic of probereduction and the metabolic activity of anaerobic sludge (Chenet al., 2015). As shown in Fig. 2(b), the biodegradation kineticswere studied and showed that the biodegradability of sludge wasdifferent. More precisely, the metabolic activity of inoculum wasdifferent for each sludge. The explanation is that the behavior ofinoculum was dependent on its ability to break down the organicmatter according to classes of biodegradability. For example, the

Please cite this article in press as: Bellaton, S., et al. Early assessment of a rapidage sludge. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.201

primary sludge was rapidly biodegraded compared to biologicalsludge. Indeed, biological sludge is mainly composed of complexmolecules. Therefore, these outcomes demonstrate the interestand the relevance of this rapid test in order to quickly assess theanaerobic biodegradability of sewage sludge in only 2 days. Never-theless, the control (blanks) curves showed an extended lag phase.According to Wang et al. (2015b), an inoculum pre-incubation at37 �C and a filtration of inoculum introduce a lag-time and by

alternative method for the estimation of the biomethane potential of sew-6.01.139

Fig. 2. Typical fluorescence curves (a) and biodegradation kinetics (b) of sewage sludge under anaerobic conditions. Control: endogenous metabolic activity of inoculum.

4 S. Bellaton et al. / Bioresource Technology xxx (2016) xxx–xxx

consequently a lowest enzyme activity and a lowest methaneyield. The extended lag phase in control sample confirmed theimpact of pre-treatments on enzyme activity.

3.2. Methane production: Envital� kit and AMPTS

Envital� gives an estimation of methane production by an indi-rect measure of the organic matter biodegradability based onmicrobial activity. The operational performance of the Envital�

Please cite this article in press as: Bellaton, S., et al. Early assessment of a rapidage sludge. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.201

technology was evaluated thanks to the estimation of biomethaneproduced with 1 g of sodium acetate, knowing that the theoreticalbiomethane potential is of 373 NmL CH4/g VS. The estimated bio-methane of 1 g of sodium acetate has been maintained as controlin the BMP test. The estimated result was 319 NmL CH4/g VS. Fur-thermore, the values of the standard deviation of repeatabilitywere satisfying, below 10% , like the AMPTS method. These resultssuggested that the Envital� technology was reliable to estimate themethane production. The comparison between a direct measure

alternative method for the estimation of the biomethane potential of sew-6.01.139

Fig. 3. Comparison between AMPTS and Envital� on BMP parameter. PS (Primary Sludge); BS-1 to BS-4 (Biological Sludge – Tank); BS-5 to BS-8 (Biological Sludge-Biofilter);MS (Mixed Sludge); TS (Tertiary Sludge). ⁄Same inoculum for AMPTS and Envital�.

S. Bellaton et al. / Bioresource Technology xxx (2016) xxx–xxx 5

with AMPTS� and an indirect measure with Envital� on the BMPparameter was investigated on five experiments with a total ofnineteen samples of sewage sludge. The methane production withthe Envital� technology was estimated in 2 days while AMPTSrequired 21 days. The endogenous methane produced, correspond-ing to the anaerobic sludge used as inoculum, was subtracted forthe other samples. The AMPTS measurements were used as refer-ence for comparison with the alternative method. An uncertaintylimit of 25% usually admitted (Lesteur et al., 2011; Pham et al.,2013) was chosen in order to validate BMP estimated by Envitaltechnology against the reference method. Furthermore, the BMPof acetate measured with the AMPTS was 251 CH4/g VS being67% of the theoretical biomethane potential thus an uncertaintylimit of 25% was justified. Fig. 3 presents the methane productionfor the nineteen samples with both technologies. For the samplesidentified by (⁄), the inoculum used was collected the same dayfor both analysis. Results achieved seemed to be in the same orderof magnitude between two methods and, with the literature(Carrère et al., 2010; Jensen et al., 2014) except for one sample,the tertiary sludge. Indeed, the estimated values with the alterna-tive assay were in the uncertainty range except for this sample.These first outcomes confirm that the surrogate method can besuccessfully correlated with the sludge biodegradability in ADand can estimate the biomethane production of municipal sludgein less than 48 h. A hypothesis for the tertiary sludge sample wasthat the inoculum used was different. According to Strömberget al. (2014), it seems that this is the origin of inoculum whichcould have an impact on the biomethane production. For that mat-ter, the biomethane production is a function of microorganismgrowth (Nopharatana et al., 2007) suggesting thus the relevanceto use a fluorescent probe, which measures the metabolic activityof microorganisms, as an alternative method to assess thebiodegradability in AD.

All results suggest that the Envital� technology can be a rapid,high-throughput and relevant alternative approach in order tocharacterize the biodegradability of sewage sludge in AD and toquickly answer operational requests. With this fast assay manyaspects of AD could be investigated, such as inhibition phenomena,pre-treatment, or nutrient supplementation.

Please cite this article in press as: Bellaton, S., et al. Early assessment of a rapidage sludge. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.201

4. Conclusions

This rapid analysis based on fluorescence measurement allowedthe estimation of the biodegradation kinetics and the biomethaneproduction of sewage sludge in only 2 days. The obtained resultswere comparable with measurements achieved by AMTPS. Basedon these first results, future works will investigate the relevanceof these two methods in order to quickly answer different prob-lematics such as toxicity, the impact of inoculum (origin, pre-treatment), or biological process diagnosis in synergy with othertechnologies (spectroscopy, modeling).

Acknowledgements

The authors are grateful to the research program ‘‘MOCOPEE”(MOdeling Control and Optimization of wastewater treatment Pro-cEssEs) for its technical and financial support.

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