Evaluation of the effects of low energetic microwave irradiation on anaerobic digestion

Journal of Environmental Management 202 (2017) 69e83

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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman

Research article

Evaluation of the effects of low energetic microwave irradiation on anaerobic digestion Bert Bastiaens, Rob Van den Broeck, Lise Appels, Raf Dewil* KU Leuven, Department of Chemical Engineering, Process and Environmental Technology Lab, J. De Nayerlaan 5, B-2860, Sint-Katelijne-Waver, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2017 Accepted 27 June 2017

The present study investigates the effects of microwave irradiation on the performance of anaerobic digestion processes. A first set of experiments is performed to distinguish the upper limit of the applied energy levels. Secondly, the effects of these treatments on the performance of the digestion process are evaluated in 3 experimental setups: (i) monitoring the acetic acid degradation, (ii) performing a biological methane potential (BMP) assay and (iii) conducting a specific methanogenic activity (SMA) test. The solubilisation experiment reveals a limited degree of disintegration of anaerobic biomass up to a microwave treatment of 10000 kJ/kg TS. Above this threshold value the soluble COD level started to rise, with up to 350% at 30000 kJ/kg TS regardless of the microwave output power. Because solubilisation of the biomass increases the easily degradable content, this would lead to false observations regarding increased activity. Therefore, solubilisation is minimized by limiting the microwave treatment to a maximum of 6000 kJ/kg TS during the second part of the experiments. Monitoring the degradation of acetic acid after a low intensity microwave treatment, reveals that microwave irradiation shortens the lag phase, e.g., from 21 to 3 h after a microwave treatment of 1000 kJ/kg TS at 100 W. However most treatments also result in a decrease of the maximum degradation and of the degradation rate of acetic acid. BMP assays are performed to evaluate the activity and performance of the entire anaerobic community. Every treatment results in a decreased biogas production potential and decreased biogas production rate. Moreover, each treatment induced an increase of the lag phase. The SMA experiments show no influence of the microwave irradiation in terms of biogas or methane production. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic digestion Biogas Microwaves Stimulation Specific methanogenic activity

1. Introduction The increasing concern on climate change and the increasing energy demand have stimulated the search and application of renewable energy sources (Office for official publications of the European Communities, 2009). Anaerobic digestion of biomass and other organic waste streams proves to be a very interesting process in the search for durable and renewable energy (Appels et al., 2008). It is a microbiological process in which complex substrates such as waste activated sludge and food waste are degraded to biogas. The degradation process consists of 4 subsequent steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis, each performed by a different microbial community. During methanogenesis, methane is produced through the degradation of acetate and the combination of hydrogen and carbon dioxide. Advantages such as volume reduction, the reduction of pathogens and the valorization of waste * Corresponding author. E-mail address: [email protected] (R. Dewil). http://dx.doi.org/10.1016/j.jenvman.2017.06.062 0301-4797/© 2017 Elsevier Ltd. All rights reserved.

by producing methane indicate that it is a promising technique to produce a renewable fuel. However, drawbacks such as long retention times and an organics reduction of only 50e60% ask for a further efficiency increase of this technology (Bolzonella et al., 2005; Metcalf and Eddy, 2003). The most important approach currently under investigation is the application of a pre-treatment to increase the biodegradability of the substrate. These pre-treatments are often characterized by high energy requirements (Ariunbaatar et al., 2014; Harris and McCabe, 2015). The present study is focused on a novel approach, i.e., the use of a low energetic treatment of the anaerobic biomass to stimulate its activity within the bioreactor itself. Instead of focusing on the substrate, this approach treats the anaerobic microbial community to investigate the effects on its performance. Some previous literature sources suggest the beneficial effects of microwaves on different types of micro-organisms. For example, previous research reported an increase in cell size, cell growth and methane production after irradiating pure cultures of Methanosarcina barkeri DSM-804 with microwaves (Banik et al., 2006). Some experiments involving the microwave irradiation of an

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anaerobic microbial community report an increase in biogas production and an increased diversity of the Archaea present in the reactors (Cydzik-Kwiatkowska et al., 2012; Jang and Ahn, 2013;
ski et al., 2007). Zielin The goal of this paper is to assess the effects of a low energetic microwave treatment on the performance of the anaerobic microbial community. In the first part, the solubilisation of the anaerobic biomass through microwave irradiation is investigated to select the applicable, preferably non-lethal, energy levels. Subsequently, the influence of a microwave treatment on the performance of an anaerobic community is investigated. First, anaerobic sludge samples are treated using microwave irradiation and are subsequently fed with acetic acid. The degradation is monitored and is used as a measure for the activity of the methanogenic community. Secondly, a biological methane potential (BMP) assay is used to monitor the effects of a microwave treatment on anaerobic digestion. Finally, a specific methanogenic activity (SMA) experiment is performed to assess the effects of microwave irradiation on the activity of the methanogenic community. 2. Materials and methods 2.1. Anaerobic biomass Digestate samples are taken from the full-scale sludge digester located at the municipal wastewater treatment plant of Aquafin Antwerpen-Zuid (Belgium). The main characteristics of the biomass are included in Table 1. 2.2. Microwave treatment The microwave treatment is applied using a Monowave 300 microwave synthesis reactor (Anton-Paar). The Monowave 300 is a laboratory scale microwave reactor, generating a continuous (i.e., non-pulsed) microwave field with a variable output power between 1 and 850 W. The setup ensures the homogeneous transfer of energy to the complete reaction volume in the vessel. All experiments are performed using 30 mL borosilicate vials and are filled with 20 mL of digestate. The system offers the possibility to fully control the reaction conditions including power, pressure and temperature. Maximum operation pressure and temperature are 30 bar and 300
C, respectively. During the microwave treatment, the digestate is stirred using a magnetic stirring bar at 600 rpm to ensure the homogeneity of the sample during the microwave treatment. For a chosen specific energy (SE), the required microwave irradiation time is calculated according to Equation (1):

tMW ¼

SE*TS*msludge P

(1)

With tMW ¼ microwave irradiation time [s], SE ¼ specific energy [kJ/kg TS], TS ¼ total solids content of the anaerobic sludge [g TS/g sludge], msludge ¼ amount of sludge [g] and P ¼ output power [W] (Kuglarz et al., 2013; Tang et al., 2010).

Table 1 Characteristics of the biomass taken from the full-scale anaerobic digester of Aquafin Antwerpen-Zuid. Total Solids (TS) [g TS/kg sludge] Volatile Solids (VS) [g VS/kg sludge] pH Conductivity at 25
C [mS/cm] Total COD (tCOD) [mg O2/L] Soluble COD (sCOD) [mg O2/L]

46.184 26.572 7.88 12.57 46000 2459

± ± ± ± ± ±

0.369 0.371 0.16 1.88 525 194

2.3. Experimental setup 2.3.1. Solubilisation In order to investigate the solubilisation of anaerobic biomass, a 30 mL reaction vial is filled with 20 mL (~20 g) of anaerobic biomass and placed inside the Monowave 300 cavity. The samples are subjected to microwave treatments ranging between 0 and 30000 kJ/kg TS at 3 levels of output power: 100, 475 and 850 W (corresponding to 5, 23.75 and 42.5 W/g, respectively). The required irradiation time is calculated according Equation (1). During the treatment, both temperature and pressure inside the reaction vial are monitored. After the treatment, the vial is cooled down rapidly in an ice bath to prevent further thermal effects. Following the cooldown the treated biomass is analyzed for soluble COD (sCOD), electrical conductivity (EC), pH and ionic content (IC). Each experiment is performed in triplicate in order to evaluate the repeatability and reproducibility of the process. 2.3.2. Acetic acid degradation The influence of the microwave treatment on the activity of the methanogens is evaluated by monitoring the degradation of acetic acid (HAc). Based on the results of the solubilisation experiments, SE levels between 0 and 6000 kJ/kg TS are applied for these tests, following the procedure already described in section 2.3.1. Each test is carried out in triplicate to enable a statistical comparison. After the microwave treatment, the sludge sample (20 mL) is transferred into a 50 mL reaction vial. Subsequently, it is fed with 20 mL of a 2 g/L HAc solution. Thereby a concentration of 1 g/L acetic acid is obtained, which is suggested to be the optimal concentration to evaluate the acetoclastic methanogenic activity (Angelidaki et al., 2009). Subsequently, the reaction mixture is distributed over 8 small reaction tubes (3.5 mL) and all the tubes are placed in an incubator at 37
C. The reaction volume is distributed over the smaller tubes to prevent contact with oxygen by frequently opening the reaction tube for sampling. Therefore, each tube represents 1 sampling point. Sample frequency is high during the first 12 h to enable the determination of the lag phase. The acetic acid content of each sample is determined using gas chromatography with a flame ionization detector (GC-FID). In order to compare the different datasets, the data are presented as a degradation profile. Degradation is defined as the fraction of acetic acid that is consumed, calculated using Equation (2).

Degradation ¼

½HAc0   ½HAct  ½HAc0 

(2)

With [HAc0] ¼ acetic acid concentration at start (t ¼ 0 h) and [HAct] ¼ acetic acid concentration at time t. To quantify the results obtained by monitoring the degradation as a function of time the profiles are fitted using the modified Gompertz equation, presented in Equation (3). The modelling of the curves is performed using the Solver add-in of Microsoft Excel 2013.


  Rm *e *ðl  1Þ þ 1 M ¼ P*exp  exp P

(3)

With M ¼ observed degradation, P ¼ degradation potential, Rm ¼ degradation rate [h1] and l ¼ the lag phase [h] (Patil et al., 2012; Zhen et al., 2015). 2.3.3. Biological methane potential In a second experiment, the effects of low-level microwave irradiation on the performance of anaerobic digestion is investigated through a biological methane potential assay (BMP)

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(Angelidaki et al., 2009; Angelidaki and Sanders, 2004). In accordance with the experiment regarding the degradation of acetic acid, the same 5 microwave treatment conditions are applied to the anaerobic biomass. The BMP assays are conducted using 50 mL serum vials with crimp caps and septa. Treated sludge samples (20 mL) are mixed with a synthetic waste (Table 2) in a substrate to inoculum ratio (S/I) of 0.5 on VS-basis to a working volume of 40 mL. Biogas production is monitored daily using the water displacement method. Acidified water is used to prevent solubilisation of carbon dioxide gas. The composition of the biogas is analyzed using a gas chromatograph equipped with a thermal conductivity detector (GC-TCD). In order to compare the different biogas production profiles, the data sets are fitted using a model to determine kinetic parameters. Both the modified Gompertz model (Equation (3)) and the Richards equation (Equation (4)) are evaluated using the F-test, otherwise known as the extra sum-of-squares F-test (Archontoulis and Miguez, 2015; Zwietering et al., 1990)

    n hm io 1y 1þ1y m *ð1 þ yÞ *ðl  tÞ y ¼ A* 1 þ n*expð1 þ yÞ*exp A (4) With y ¼ cumulative biogas production [mL/g CODadded], A ¼ maximum biogas potential [mL/g CODadded], mm ¼ maximum biogas production rate [mL/g CODadded.d], l ¼ lag time [days] and n ¼ shape factor (n ¼ 0: modified Gompertz model, n ¼ 1: logistics model) (Birch, 1999). Equation (4) presents the Richards equation, it is clear that it is more complex than the modified Gompertz equation (Equation (3)) due to an extra parameter, i.e. the shape factor. An important drawback of the modified Gompertz equation is the fixed position of the inflection point, i.e. at 1/e of the maximum biogas potential P (Archontoulis and Miguez, 2015), therefore limiting the use of the modified Gompertz equation for non-ideal gas profiles, e.g. stepwise biogas production profiles. In the Richards equation, the location of the point of inflection is determined by the shape factor n, giving the model more flexibility regarding asymmetric growth-/production profiles. However, this shape factor has no (micro-)biological meaning (Annadurai et al., 2000; Archontoulis and Miguez, 2015).

2.3.4. Specific methanogenic activity A specific methanogenic activity (SMA) assay is used to investigate, determine and quantify the activity of the methanogenic Archaea within the anaerobic community (Cho et al., 2005; Souto et al., 2010). Again, the effects of a microwave treatment ranging from 0 to 6000 kJ/kg TS are investigated. The sludge samples are fed with 1 g/L sodium acetate (NaAc) to a 50 mL serum vial (Angelidaki et al., 2009). The biogas production is monitored using the water displacement method and the methane

Table 2 Composition and characteristics of the synthetic feed. Cornstarch Baby milk powder Yeast extract NH4Cl MgSO4$7H2O CaCl2$2H2O CaCO3 COD TN TS VS

17.44 20.78 2.81 1.55 3.14 0.54 0.17 50 1 40 37

g/L g/L g/L g/L g/L g/L g/L g/L g/L g TS/kg synthetic feed g TS/kg synthetic feed

71

content is analyzed using GC-TCD. Every experiment is performed in triplicate to enable statistical comparison. Based on the results of a F-test, the best suited model (i.e. modified Gompertz or Richards equation) is chosen to fit the biogas production to quantify the methanogenic activity. 2.4. Analytical methods The sCOD is measured using COD test kits and a DR3900 spectrophotometer (Hach). For both sCOD and IC the samples are subsequently centrifuged (10 min  14500 rpm) and filtrated (Whatman 934-AH, 1.5 mm). The conductivity is measured using a HQ30d digital multimeter (Hach) equipped with a conductivity sensor, calibrated with a 1000 mS/cm sodium chloride standard solution. The pH is measured using a WTW pH meter combined with a SI-analytics pH sensor calibrated with technical buffers of pH 4 and 7. TS and VS content are determined according to the standard methods (Greenberg and Eaton, 1999). 2.4.1. Ion chromatography The ionic content is analyzed using a Dionex ICS-1000 Ion Chromatography System equipped with a cation exchange membrane and methanesulfonic acid (20 mM) as eluent. The chromatograph operates at a pressure of 1200 psi, a suppressor current of 59 mA and a temperature of 30
C. A 20-fold dilution is applied to prevent the clogging of the filter in the autosampler of the equipment. 2.4.2. GC-FID Volatile fatty acid (VFA) concentrations are determined using an Agilent A7890 gas chromatograph equipped with a flame ionization detector and a HP-FFAP capillary column (30 m  250 mm x 0.25 mm). Helium is used as carrier gas at a flowrate of 1.5 mL/min and a split ratio of 30:1. The injector, oven, detector temperature and temperature ramp are 200
C, 45
C, 240
C and 10
C/min during 10 min, respectively. The volatile fatty acids are extracted from the sludge samples using sodium chloride, sulphuric acid and diethyl ether. The VFA concentrations are determined using an external calibration curve from 125 to 4000 ppm for 8 VFAs, respectively acetic acid (HAc), propionic acid (HProp), butyric acid (HBut), isobutyric acid (HIsobut), valeric acid (HVal), isovaleric acid (HIsoval), caproic acid (HCap) and levulinic acid (HLev). 2-Methylvaleric acid is used as the internal standard to account for the extraction efficiency. The software module Chemstation (Agilent) is used to process the results. 2.4.3. GC-TCD The composition of the biogas is measured using a HP 6890 gas chromatograph equipped with a thermal conductivity detector. A Poraplot-U capillary column (25 m  0.53 mm x 20 mm) is used to separate the components with helium as the carrier gas at a total flowrate of 179.0 mL/min and a split ratio of 50:1. The injector, the oven and the detector operate at respectively 180
C, 80
C and 200
C. Samples of 500 mL are injected manually using a gastight syringe. Methane and carbon dioxide content are determined using the Chemstation software (Agilent). 2.5. Statistical analysis All experiments are performed in triplicate to calculate a statistically correct mean and standard deviation. 2.5.1. F-test In order to determine which model best suits the obtained biogas production profiles, the fit acquired by modified Gompertz is

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compared to the Richards equation. Since both equations are ‘nested’, i.e. related to each other (modified Gompertz ¼ Richards equation with n ¼ 1), the extra sum-of-squares test (i.e., the F-test) can be performed. Based on the (minimum) sum-of-squares value and the degrees of freedom of both equations an F-factor is calculated according to Equation (5). Finally, this F-value is used to calculate the P-value. The accuracy and applicability of both fits are evaluated based on the P-value (Motulsky and Christopoulos, 2003).



. SSRichards . F¼ DFmod:gomp  DFRichards DFRichards SSmod: gomp  SSRichards

(5)

With SSmod.gomp ¼ sum-of-squares obtained using modified Gompertz equation, SSRichards ¼ sum-of-squares value acquired by the Richards equation, DFmod;Gomp ¼ number of degrees when using modified Gompertz to fit the dataset and DFRichards ¼ the number of degrees when fitting the data-points with the Richards equation. 3. Results and discussion 3.1. Solubilisation In accordance with the literature, the soluble fraction of the chemical oxygen demand (sCOD) is chosen as the main output parameter in the solubilisation experiments (Hu et al., 2012; Serrano et al., 2016). Fig. 1 presents the solubilisation as a function of applied SE, expressed as the increase in sCOD compared to the blank in Fig. 1A and expressed as the ratio between sCOD and tCOD in Fig. 1B. The results indicate no significant increase of sCOD up to 6000 kJ/kg TS for all applied output power levels. At 8000 kJ/kg TS the average increase is 5%. Starting from 10000 kJ/kg TS the soluble fraction increases more rapidly: from an increase of 15% at 10000 kJ/kg TS to 270e300% at 30000 kJ/kg TS. When accounting for the calculated deviations, no significant difference is observed between the treatments at different levels of output power (Fig. 1B). Since no previous papers are found on the solubilisation of anaerobic sludge by microwave irradiation, the results are compared to literature results on the microwave treatment of waste or thickened activated sludge (Table 3). At the university of Szeged the effects of different levels of output power on the solubilisation of sludge were investigated, with a maximum power output of
des et al., 2011). These results show no significant 700 W (Besze differences between the power levels: sCOD levels increase by
des 300% at 6000 kJ/kg TS, independent of the output power (Besze

Table 3 Literature overview: solubilisation of waste activated sludge with microwave irradiation, expressed as the increase of soluble COD compared to the blank. P [W]

SE [kJ/kg TS]

sCOD increase [%]

References

700 700 400 800 250 500 800 400 700

9800 29000 4412 7006 6000 6000 8235 20000 20000

400 950 350 430 300 300 207 25 215

(Ahn et al., 2009) (Ahn et al., 2009) (Tang et al., 2010) (Tang et al., 2010)
des et al., 2011) (Besze
des et al., 2011) (Besze (Appels et al., 2013) (Serrano et al., 2016) (Serrano et al., 2016)

et al., 2011). The same conclusion can be drawn based on the results acquired by Kuglarz and co-workers, who determine the COD solubilisation percentage after achieving a temperature of 70
C in 1L of sludge with both 700 W and 900 W output power, and observe an equal increase of sCOD percentage from 1 to 32%. These observations confirm the results presented in Fig. 1: the increase in sCOD appears to be independent of the microwave output power (Kuglarz et al., 2013). However, at the university of Cordoba a pilot scale microwave setup was used to observe an increase of the sCOD of 25% after a treatment of 20000 kJ/kg TS at 400 W and an increase of about 220% after a treatment of 20000 kJ/kg TS at 700 W. These results indicate that the level of solubilisation depends on the level of microwave output power. But it has to be noted that the above mentioned treatments are performed on different sludge samples with significantly different initial levels of sCOD: 8625 and 1835 for the 400 and 700 W treatment, respectively. Therefore the results cannot be compared and no conclusion can be drawn regarding the relationship between the initial sCOD value and the increase in sCOD due to microwave treatment (Serrano et al., 2016). Comparing the results presented in Fig. 1 to the values shown in Table 3, it is clear that more energy is required for the solubilisation of anaerobic sludge compared to aerobic sludge. No significant increase in the sCOD level of anaerobic sludge is observed up to a treatment of 10000 kJ/kg TS, where literature on aerobic sludge reports increases in sCOD of aerobic sludge up to 400% at those energy levels (Table 3, Fig. 1). Similarly, at high energy levels such as 30000 kJ/kg TS, an increase of sCOD by about 350% is observed during the experiments (Fig. 1), where the literature reports increases of sCOD levels of aerobic sludge by up to 950% (Table 3). These observations indicate that an anaerobic microbial community is more resistant to microwave treatment compared to an aerobic community.

Fig. 1. Anaerobic sludge solubilisation as a function of SE with microwave irradiation at 3 different levels of output power: A) relative increase of sCOD compared to blank, B) sCOD compared to tCOD.

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In previous studies evaluating the effects of microwave radiation on pure microbial cultures, electrolyte leakage is used to evaluate the condition of the cell membranes of the microorganisms (Campanha et al., 2007; Fang et al., 2011). Breaking or opening the cell membranes results in the release of intracellular material (e.g. salts, organic acids, …) to the environment, thereby influencing the conductivity of the sludge. Some papers report a reversible leakage of electrolytes due to microwave irradiation of cells. These kind of observations have led to the hypothesis that low energy microwave irradiation creates temporary (reversible) pores in the cell membranes (Shamis et al., 2012, 2011). To investigate the electrolyte leakage due to the solubilisation of anaerobic sludge, conductivity, pH and concentrations of most relevant cations þ 2þ (Naþ, NHþ and Ca2þ) are measured. 4 , K , Mg Fig. 2 presents the results of monitoring both pH and EC. The microwave treatment induces no change in pH, regardless of SE and output power (Fig. 2A). At first glance, the microwave treatment affects the EC of the biomass (Fig. 2b). Up to 10000 kJ/kg TS no significant effect on the conductivity is observed, regardless of the applied output power. A further increase of the specific energy appears to induce a decrease of the conductivity: up to 20% after 30000 kJ/kg TS. However, accounting for the calculated deviations there is no significant difference in the EC of the control samples and this of the treated samples. This observation is in contradiction with previous literature findings. Fang and co-workers report an increase in conductivity as a measure for electrolyte leakage by 100% when subjecting a culture of Aspergillus parasiticus to a temperature increase to 70
C by both microwave and conventional heating (Fang et al., 2011). Campanha and co-workers also reveal that microwave irradiation (650 W) damages the integrity of the cell membrane of a pure culture of Candida albicans (Campanha et al., 2007). Research investigating the effects on different types of cell membranes observes that microwave irradiation (900 W) induces lethal effects on pure cultures of both gram negative and gram positive bacteria (Gedikli et al., 2008). These distinct scientific reports confirm that microwave irradiation induces an increase of the electrolyte concentration of a microbial suspension. However, it should be noted that the abovementioned literature performed experiments on pure cultures, whereas the research reported in this paper is performed on a mixed culture. þ 2þ The release of Naþ, NHþ and Ca2þ to the liquid phase 4 , K , Mg by the microwave treatments is depicted in Fig. 3. Based on the evolution of the conductivity in Fig. 2, a similar profile is expected for some of the cations. Indeed, Fig. 3 depicts no significant changes in concentration of either of these elements. These findings are not in line with observations reported by other researchers. Ahn and

73

co-workers, for example, investigate the solubilisation of municipal secondary sludge and report an increase of the Ca2þ-concentration by 13% after a microwave treatment of 29000 kJ/kg TS at 700 W (Ahn et al., 2009). Moreover, at the lab of Bohdziewicz, the concentration of Ca2þ and Mg2þ are monitored to evaluate the effect of microwave irradiation on floc structure of waste activated sludge. Both microwave output power (700 W and 900 W) and irradiation time (0e8 min) are varied in the experiment. No significant influence of the output power level is observed although the levels of both Ca2þ and Mg2þ increase with the irradiation time: a maximum increase of þ92% and þ170% for Ca2þ and Mg2þ, respectively, after 8 min of microwave treatment (Bohdziewicz et al., 2011). While the abovementioned literature sources conclude that microwave irradiation of sludge induces leakage of ions, accompanied by an increase in electrical conductivity by microwave irradiation, the results of this study suggest otherwise. It is important to note that the composition of anaerobic sludge is completely different from secondary sludge, which could lead to the opposite observations
ska, 2016; Narihiro and and results (Cydzik-Kwiatkowska and Zielin Sekiguchi, 2007). For example, the conductivity of secondary sludge ranges from about 1000 to 2000 mS/cm, while the results presented in Fig. 2 reveal an initial conductivity of about 12 mS/cm (Shafei et al., 2005). This shows that the conductivity of anaerobic sludge exceeds this of aerobic sludge by a factor 10. So, in order to observe a significant change of the electrical conductivity, a much larger amount has to be solubilized in anaerobic sludge compared to its aerobic counterpart. The large measurement uncertainties (standard deviations) in Fig. 3 can be explained by the high dilution rate required for the analysis. The concentration in the dilutions may vary due to inhomogeneities in the original sludge samples. Another important remark to take into consideration is that the high initial cation concentrations can obscure the release of low amounts of cations. This would lead to false conclusions regarding the electrolyte leakage. 3.2. Acetic acid degradation The degradation of acetic acid is monitored to investigate the effects of low energy microwave radiation on the performance of the methanogenic community. To ensure minimal solubilisation, a maximum microwave treatment of 6000 kJ/kg TS is chosen based on the results in Fig. 1. The evolution of the acetate concentration during the acetic acid degradation experiment is presented in Fig. 4. The represented data points are the average of 3 replicates, the standard deviations vary between 5 and 10%. The parameters obtained by the modified Gompertz equation are shown in Table 4.

Fig. 2. A) pH and B) Electrical conductivity (EC) at 25
C versus specific energy of microwave irradiation of anaerobic sludge to assess solubilisation.

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þ 2þ Fig. 3. Evolution of the cation concentrations after microwave treatment: A) Naþ, B) NHþ and E) Ca2þ. 4 , C) K , D) Mg

As illustrated in Fig. 4, various profiles show an increase of the acetic acid concentration during the first 12 h. Calculations resulted in a standard deviation of about 0.49, i.e. 49%, for those data points. This deviation is larger than the average value for degradation of the associated replicates. Therefore, it is proposed that an observed increase of acetic acid can be neglected. Alternatively, it is possible that a small amount of acetate is in situ produced from residual organic material that is present in the digestate or is released by the microwave treatment. At an output power of 100 W the fitted curves clearly indicate that the microwave irradiation positively affects the lag phase of the acetic acid degradation (Fig. 4A). A 1000 kJ/kg TS treatment at 100 W induces the shortest lag phase: only 3 h compared to 24 h for the control reactors. But it is noticed that the fitted curve of 1000 kJ/ kg TS shows an offset at sampling time t ¼ 0 h. Secondly, the fitted

curve does not show any flattening although all the added acetic acid has been consumed. This is caused by the absence of a sampling point between 24 and 48 h or a point over 48 h. According to Table 4 the predicted maximum degradation corresponding to this fit is 6.157 (marked in bold). By adding the extra restraint of “P  1”, this impossible value is avoided. The results of the latter fit are indicated using the symbol “*” in Table 4 and Fig. 4A. Up to the sample point of 24 h, both curves for 1000 kJ/kg TS at 100 W are equally good. But after taking into account the deviation of the fit to the sampling point after 48 h (, symbol), the first fitting is considered the best. Due to the stepwise shape of the degradation profile, the modified Gompertz could not fit the degradation at 1000 kJ/kg TS and 100 W correctly. The fitted curves obtained by the treatments of 2000 and 6000 kJ/kg TS at 100 W overlap during the first 10e15 h, indicating

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Fig. 4. Acetic acid degradation during 48 h after a microwave treatment: A) 100 W, B) 475 W and C) 850 W, fitted using the modified Gompertz equation.

a similar lag phase for both treatments. But the responses of the fittings show that, for an output power of 100 W, the lag phase at 2000 kJ/kg TS is almost twice as long compared to 6000 kJ/kg TS: respectively 9.6 and 5.2 h (Table 4). This anomaly is caused by the definition of the modified Gompertz equation. According to Equation (3), the inflection point of the model corresponds to the time after which the gas production reaches about 36.8% (1/e) of the maximum cumulative production (Birch, 1999; Goshu, 2013). After about 20 h, the degradation rate of the 6000 kJ/kg TS treated sludge

decreases. This results in a significantly lower maximum degradation compared to the blank and 2000 kJ/kg TS profiles. Since the maximum degradation is reached much earlier, the corresponding lag phase is much shorter. In Fig. 4A the smallest decrease in the lag phase is observed in the 4000 kJ/kg TS test tubes, i.e., 16.6 h compared to 21.4 h, but the average degradation rate appears to approach this of the 2000 kJ/kg TS tubes. Regarding the degradation of acetic acid after 48 h, only the treatments of 1000 and 2000 kJ/kg TS approximate the blank (Fig. 4A). The test tubes treated with

Table 4 Modelling parameters using modified Gompertz to fit acetic acid degradation by anaerobic sludge after a microwave treatment, * ¼ extra constraint ‘P  1’. SE [kJ/kg TS]

0 1000 1000* 2000 4000 6000

100 W

475 W

850 W

Max. degradation [(C0-C)/C0]

l [h]

Max. degradation [(C0-C)/C0]

l [h]

Max. degradation [(C0-C)/C0]

l [h]

0.971 6.157 1.000 1.032 0.776 0.461

21.4 26.0 3.0 9.6 16.3 5.2

1.005 0.922 n/a 0.939 0.440 0.296

17.3 18.7 n/a 7.6 17.5 1.5

0.974 1.207 n/a 0.916 0.835 0.239

21.3 0.0 n/a 7.6 19.1 15.0

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Table 5 Observed acetic acid degradation after 48 h, based on GC-FID analysis and, expressed as %degraded. SE [kJ/kg TS]

100 W

0 1000 2000 4000 6000

94.9 100 85.5 67.4 43.6

± ± ± ± ±

475 W 6.45 0.00 5.69 9.37 2.09

97.8 91.8 78.4 40.5 29.1

± ± ± ± ±

850 W 3.73 0.0142 08.46 1.09 5.93

94.9 84.5 84.4 83.2 23.9

± ± ± ± ±

6.45 3.08 4.48 11.2 3.69

4000 and 6000 kJ/kg TS show a significant decrease in acetic acid degradation. The numerical values observed at the sample point of 48 h are represented in Table 5. For the treatment of 100 W, only about 44% of the acetic acid is degraded in the 6000 kJ/kg TS treated test tubes. It appears that an increase of the specific energy level induces a systematic decrease of the maximum acetic acid degradation as shown in Fig. 5. Starting from 1000 kJ/kg TS at 100 W output power the degradation after 48 h decreases almost linearly with an increasing specific energy level (R2 ¼ 0.9948). An evaluation of the results of the VFA analysis showed that most of the samples only contained acetic acid. At the 100 W treatment, only the test vials of 6000 kJ/kg TS showed traces of propionic, isobutyric and isovaleric acid. The results presented in Fig. 6 clearly show the appearance of these VFAs, 9 h after the start of the experiment. An accumulation of VFAs other than acetic acid could indicate that both acetogenesis and methanogenesis are inhibited by the treatment, resulting in failure of the anaerobic system. But the concentration of most of the VFAs does not rise above 4 ppm. Only propionic acid appears at a concentration of

12 ppm after 24 h, but it has already disappeared after 48 h. Propionic acid is known to inhibit the digestion process (Gerardi, 2003; Labatut and Gooch, 2012; Wang et al., 2009) but the observed concentrations are close to the detection limit of the GC, and therefore their influence on the degradation of acetic acid can be neglected. Based on these results, it can be concluded that for a treatment at 100 W an increase in the specific energy level induces a decrease of the lag phase, but also induces a decrease of the average degradation rate since the blank has the highest degradation after 48 h (see Table 5). The degradation profiles obtained by treating the anaerobic biomass with 475 W do not resemble those of the 100 W treatment as seen in Fig. 4B. In this case the profile of 1000 kJ/kg TS almost perfectly overlaps with the degradation observed in the blank tubes. This is confirmed by the values generated by fitting the modified Gompertz equation and the degradation after 48 h (see Fig. 4 and Table 5). The treatment of 4000 kJ/kg TS also shows no effect on the lag phase of the degradation. But in contrast with the 1000 kJ/kg TS treatment, a lower degradation is observed for these test tubes. Both the predicted maximum degradation and the observed degradation are about 60% lower compared to the blank. The treatment of 2000 kJ/kg TS induces a decrease in the lag phase of the process from 17.3 to 7.6 h. As for the predicted maximum degradation, 2000 kJ/kg TS is a very interesting treatment, because still 93.9% of the acetic acid is degraded. Considering the natural variation for microbiological processes, there is no significant difference between both treatments. But by evaluating the degradation after 48 h it is clear that although 2000 kJ/kg TS induces a significant decrease in lag phase, the degradation rate is much lower. After 48 h only about 78% of the acetic acid is degraded,

Fig. 5. Overview of the degradation observed 48 h after a MW treatment: A) 100 W, B) 475 W and C) 850 W.

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Fig. 6. Monitoring VFA concentrations during 48 h after a MW treatment of 100 W, 6000 kJ/kg TS.

compared to 98% for the blank as shown in Table 5. The most remarkable observation in this experiment is the increase of acetic acid during at least 48 h after treating the sludge with 6000 kJ/kg TS: the acetic acid concentration increases with almost 30% over 48 h. Standard deviations for this profile did not exceed 6%, indicating that the increase of acetic acid is significant. Analysis of the chromatograms reveals the detection of several VFAs in the 4000 and 6000 kJ/kg TS test tubes, i.e., propionic acid, isobutyric acid, butyric acid and isovaleric acid (Fig. 7). According to the chromatograms, the VFAs appeared in the 4000 kJ/kg TS only after 9 h (Fig. 7A). Again, the concentrations are very low (<10 ppm), so no effect on the digestion process is expected. In the test tubes of the 6000 kJ/kg TS treatment, the VFAs already

Fig. 7. Monitoring VFA concentrations during 48 h after a microwave treatment of 475 W: a) 4000 kJ/kg TS, b) 6000 kJ/kg TS.

77

appeared after 5 h, with a steadily increasing concentration over the next hours (Fig. 7B). These observations indicate a reduced or inhibited activity of the methanogenesis since the decomposition of these residual intermediates in the sludge leads to an accumulation of acetic acid when the activity of the methanogenic community is decreased or completely inhibited. As a result, the pH in the reactor will decrease, possibly leading to failure of the reactor. The methanogenic microorganisms are the most sensitive organisms of the anaerobic digestion community. Their optimal pH ranges from 6.8 to 7.2 and they are also the most vulnerable when experiencing temperature changes and ammonia inhibition (Chen et al., 2008). Evaluating the degradation of acetic acid after 48 h in Fig. 5B again reveals a linear correlation (R2 ¼ 0.9618). Since a treatment of 6000 kJ/kg TS showed a production of acetic acid, it is excluded from the calculations in Fig. 5B. The results of the treatments at 850 W are depicted in Fig. 4C. The observed profiles are very similar to those found with the treatments at 100 W (see Fig. 4A). Regarding the effect on the lag phase, again it can be stated that a treatment of 1000 kJ/kg TS induces the greatest change: from 21.3 to 0 h, as presented in Table 4. Further increasing the energy decreases the improvement of the lag phase. At 6000 kJ/kg TS the lag phase is larger in comparison to the blank. A large difference with the profiles of 100 and 475 W is the degradation after 48 h and the predicted maximum degradation. Taking into account the calculated deviations there is no significant difference in the degradation of the treatments, except for the treatment of 6000 kJ/kg TS as shown in Table 5 and Fig. 4. For the latter, the degradation decreased from about 100 to 24%. No accurate trend line could be constructed in Fig. 5C since only the degradation at 6000 kJ/kg TS showed a significant deviation of the untreated test tubes (R2 ¼ 0.7314). Interpreting the results of the VFA analysis in Fig. 8, again only at 6000 kJ/kg TS other VFAs are found in the test tubes. Again, these VFAs did not appear at the start, but after 7 h in this experiment. The observed concentrations of the acids, again, are very low (<10 ppm), so the effect on the degradation process can be neglected. In general, it is observed that there is a large difference in acetate degradation between different applied output powers for the same SE level. This indicates that not the output power, but the irradiation time has a more significant effect on the performance of the methanogenic activity. Comparing the results of Figs. 1 and 5 it is observed that an increased SE level induces an increase of sCOD but decreases the degradation of acetic acid. It is not likely that the microwave treatments result in the death of microorganisms since the SE level used for the degradation experiment induced only very limited solubilisation, 4% at 6000 kJ/kg TS as shown in Fig. 1. Therefore, it is

Fig. 8. Monitoring VFA concentrations during 48 h after a microwave treatment at 850 W, 6000 kJ/kg TS.

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stated that the results indicate that low energy microwave irradiation induces an inactivation of the methanogenic community. Most of the abovementioned results indicate that a microwave treatment of anaerobic biomass has no beneficial effect on the anaerobic degradation of acetic acid. However, the observation that the microwave treatment can shorten the lag phase without negatively influencing the effective degradation can be the first step in developing an appropriate microwave treatment scheme for a continuously operated reactor setup (Fig. 4C). A possible application of such a treatment is the minimization of the acclimatization period for digesters processing a new type of feedstock or reducing the hydraulic retention times in continuous digesters, as reported by Jang and Ahn (2013). 3.3. BMP The effects of the low energy microwave irradiation on the

entire anaerobic community are evaluated using the BMP assays. In general, the Richards equation results in the best fit, except for the biogas production profiles observed after 6000 kJ/kg TS at 100 W and after 1000 kJ/kg TS at 475 W. Since it only regards 2 out of 13 profiles and since the exceedance of the significance level is minimal it is decided to fit all datasets using the Richards equation. Applying the same model to all datasets enables equal interpretation and comparison of the fitted profiles. Fig. 9 presents the observed biogas production profiles and the corresponding fits. Regardless of the applied treatment, the highest biogas production is observed at the control reactors (¼ SE 0) (Fig. 9). Considering the results for the 100 W treatment (Fig. 9A), there does not appear to be a significant difference between the different treatments, except at 100 W 1000 kJ/kg TS. As seen in Fig. 9A, a stepped profile is observed at a 1000 kJ/kg TS. The biogas production ceased after 1 day, but at day 10 the biogas production restarted to reach its end after 21 days. Such a profile indicates an

Fig. 9. Biogas production profiles of the BMP assay, fitted using the Richards equation: A) 100 W, B) 475 W and C) 850 W microwave treatment.

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79

Table 7 Maximum production rate, expressed as mL/(g CODadded.d), calculated using the Richards equations to fit the BMP profiles. SE [kJ/kg TS]

100 W

475 W

850 W

0 1000 2000 4000 6000

47 27 36 37 36

47 30 43 35 41

47 39 35 36 32

Table 8 Lag phase of the BMP assay profiles expressed in days, calculated using Richards equation.

Fig. 10. pH at the end of the BMP assay.

increased hydrolytic rate with an unmatched methanogenic rate. This unbalance results in the accumulation of organic acids, thereby prolonging the lag phase and lowering the biogas yield (Ma et al., 2015). But, as shown in Fig. 9A, the 100 W 1000 kJ/kg TS treated reactors reached a maximum production comparable to the other BMPs treated with 100 W despite the fact that the biogas production stopped from day 2 until day 10. Therefore, the 1000 kJ/kg TS expressed an increased production rate between day 10 and 21 of the digestion. Analysis of the pH of the assay revealed no significant changes and differences in pH at the end of the digestion as seen in Fig. 10. This observation indicates that no accumulation of VFA has occurred. GC-FID analysis revealed that no VFA are present in any of the reactors at the end of the digestion period (results not shown). Since the total biogas production in Fig. 9A is of the same magnitude for 1000 kJ/kg TS as for the other treatments, no significant difference in pH and VFA content at the end of the digestion is expected. As sampling is not possible during the digestion, no conclusive explanation is found for the stepped biogas production curve observed after the 100 W, 1000 kJ/kg TS microwave treatment. Taking into consideration that every BMP is performed with the same anaerobic biomass and the same synthetic waste, no rational explanation is found for the observed biogas production. After taking account of the observed standard deviations (error bars in Fig. 9), and comparing the results to the untreated samples, it can be concluded that most of the microwave treatments do not induce a significant effect on the biogas production. According to the parameters obtained using the Richards equation, presented in Tables 6e8, the untreated sludge showed the best performance. Regardless of the applied treatment, the untreated reactors have the highest maximum biogas production, the highest production rate and the shortest lag phase. Based on the biogas production curve and the corresponding standard deviations in Fig. 9, only the treatments of 6000 kJ/kg TS show some effect compared to the control reactors. At 475 W and 850 W a significant decrease in the maximum biogas production is observed, respectively 318.3 ± 26.4 mL/g CODadded and 282.5 ± 28.7 mL/g CODadded compared to 382.4 ± 21.6 mL/g

Table 6 Maximum biogas production expressed as mL/g CODadded, calculated using Richards equation on the BMP assay profiles. SE [kJ/kg TS]

100 W

475 W

850 W

0 1000 2000 4000 6000

395 344 347 338 365

395 384 340 335 320

395 332 348 357 284

SE [kJ/kg TS]

100 W

475 W

850 W

0 1000 2000 4000 6000

0.0 7.1 1.1 2.1 0.6

0.0 0.0 2.0 1.3 3.1

0.0 1.5 1.8 1.6 0.9

CODadded for the untreated samples. For the samples treated with 100 W and 6000 kJ/kg TS, a maximum biogas production of 364.0 ± 25.2 mL/g CODadded is measured. Taking account of the standard deviations, this production is not considered significantly lower than the one measured from the untreated samples. These observations are confirmed by the parameters fitted the Richards equation. Table 6 presents the predicted biogas production at time t ¼ ∞, expressed as mL biogas/(g CODadded.d). The results show that the untreated samples have the highest biogas potential. Each microwave treatment results in a lower biogas potential. The calculated maximum biogas production rates are presented in Table 7. Again, the treated samples are characterized by a lower production rate in comparison to the untreated samples. However, the treated samples all have a production rate of the same magnitude, regardless of the applied output power and specific energy. Remarkably, all the treated samples at 850 W have a similar maximum biogas production rate, but the maximum production of the samples treated with 6000 kJ/kg TS is significantly lower: only 284 mL/g CODadded compared to an average of 346 mL/g CODadded for the other samples (Table 6, Table 7). Comparing the biogas production curves (Fig. 9C), it is clear that the biogas production of the samples treated with 6000 kJ/kg TS declines starting from day 7, where the production of the other treatment has decreased from day 9. This indicates that the treatment of 850 W and 6000 kJ/kg TS inhibits the anaerobic digestion process. However, no residual VFA concentrations are observed in any of the reactors. The last biologically significant parameter derived from the Richards equation is the lag phase. The fitted values are summarized in Table 8. The fits reveal the absence of a lag phase in the production curves of the untreated samples. For the treated samples the lag phase is minimal, except for the samples treated with 100 W, 1000 kJ/kg TS microwave radiation. The Richards equation fits a lag phase of 7.2 days, corresponding to the restart of the biogas production shown in Fig. 9. The quality of the produced biogas is monitored using GC-TCD. The methane content, for every treatment is presented in Fig. 11. Since the serum vials are not flushed at the start of the experiment a considerable amount of air is present in the headspace. After 3 days the oxygen and nitrogen content has dropped to a level of ±4%. At this moment the total produced biogas amounted to 3 times the headspace volume. These results indicate that the headspace is flushed from the initial air after producing 3 times the headspace volume.

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Fig. 11. CH4-content monitored during the SMA experiment, expressed in percentage: A) 100 W, B) 475 W and C) 850 W microwave treatment.

Fig. 11 reveals that the same profile is observed at each reactor, regardless of the output power and the specific energy level. At the start of the experiment, the methane content is relatively low: about 15e20%, the corresponding CO2-concentration is 70e80%. After 13 days, the methane production reaches a plateau resulting in a concentration of about 57%, the corresponding CO2-concentration is about 40%. The remaining 3% is detected as the sum of oxygen, nitrogen and hydrogen gas. In general, the untreated samples show the best performance during the BMP experiment. Each treatment results in a decrease of both the maximum biogas production and the biogas production rate and in an increase of the lag phase. However, no effect of the microwave treatment could be observed on the methane content of the biogas. But since the biogas production decreased due to the treatment, it can be concluded that the microwave treatment decreased the methane production of the treated sludge samples. It is important to note that, although the microwave irradiation has some (negative) effects on the digestion process, no systematic effect on the biogas production could be discovered. Based on these observations it can be concluded that low level microwave irradiation of anaerobic sludge has no or a slightly negative effect on the biogas production. 3.4. SMA The SMA experiment is used to investigate the effects of low energetic microwave irradiation on the performance of the

methanogenic community. The F-test reveals that the modified Gompertz model results in a better fit of the data, the fits are added to Fig. 12. Monitoring the biogas production reveals no significant effect due to the low energetic microwave treatment. These observations are confirmed by the parameters derived from the modified Gompertz model, as presented in Tables 9e11. Using the modified Gompertz model to fit the SMA test at 100 W reveals no significant differences between the treatments as shown in Table 9. Regarding both the biogas potential and the biogas production rate the untreated samples have the best performance. But the microwave treatment of 100 W appears to have no effect on the lag phase, regardless of the specific energy level. Evaluating the modified Gompertz model for the treatments at 475 W reveals the same observations as seen at the 100 W treatment (Table 10). No significant differences are detected between the different specific energy levels and the untreated samples have the best performances in general. The parameter values that are calculated to fit the SMA profiles of the 850 W treatments are presented in Table 11. Again, no significant differences between the different specific energy levels are observed. Monitoring the biogas quality in terms of CH4-concentration reveals that each reactor has a similar methane content during the SMA experiment, regardless of the applied microwave power and specific energy level (Fig. 13). During the SMA experiments the CH4-concentration reaches its

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Fig. 12. Biogas production profiles of the SMA experiment, fitted using the modified Gompertz equation: A) 100 W, B) 475 W and C) 850 W microwave treatment.

maximum of ±42% after 2 days, CO2-concentration reached about 13%. Due to the low biogas production, the headspace is not completely flushed. Therefore, a large amount of air remained present in the headspace during the experiments. At the end of the SMA test the summed content of oxygen, nitrogen and hydrogen gas corresponds to about 45% of the available biogas.

4. Conclusions

Table 9 Fitting parameters calculated using modified Gompertz to model the SMA profile of anaerobic sludge after a microwave treatment at 100 W.

Table 10 Fitting parameters calculated using modified Gompertz to model the SMA profile of anaerobic sludge after a microwave treatment at 475 W.

The research described in this paper investigates the effects of a low energetic microwave treatment on the performance of the anaerobic digestion process. In a first step a solubilisation experiment is performed to determine the upper limit of the applied

SE [kJ/kg TS]

P [mL/g CODadded]

Rm [mL/g CODadded.h]

l [h]

SE [kJ/kg TS]

P [mL/g CODadded]

Rm[mL/g CODadded.h]

l [h]

0 1000 2000 4000 6000

332 314 309 308 299

264 234 242 238 221

0.3 0.3 0.3 0.3 0.3

0 1000 2000 4000 6000

361 328 331 330 342

237 203 198 231 228

0.2 0.2 0.2 0.3 0.3

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Table 11 Fitting parameters calculated using modified Gompertz to model the SMA profile of anaerobic sludge after a microwave treatment at 850 W. SE [kJ/kg TS]

P [mL/g CODadded]

Rm [mL/g CODadded.h]

l [h]

0 1000 2000 4000 6000

339 344 332 337 346

239 205 189 238 221

0.4 0.3 0.3 0.4 0.4

6000 kJ/kg TS. Monitoring the degradation of acetic acid reveals that microwave irradiation decreased the lag phase, but also decreases the maximal degradation of the acetic acid degradation. Since only a minimal degree of solubilisation is observed at 6000 kJ/kg TS, it can be concluded that low energetic microwave irradiation lowers the activity of the acetoclastic methanogens. The BMP assay reveals a drop in both maximum biogas production and maximum production rate after the treatments of 6000 kJ/kg TS. These results confirm the decrease of the activity

Fig. 13. CH4-content monitored during the SMA experiment after a microwave treatment: A) 100 W, B) 475 W and C) 850 W.

energy levels. Secondly, the effects of these treatments on the performance of the digestion process is evaluated in 3 experimental setups: monitoring the acetic acid degradation, performing a BMP assay and performing a SMA test. The solubilisation reveals a threshold value at a SE level of 10000 kJ/kg TS after which the sCOD levels of the anaerobic sludge samples increased significantly. The results show no difference in solubilisation due to different levels of output power. Comparing these results to the literature regarding the disintegration of waste or thickened activated sludge, it is clear that anaerobic sludge is less sensitive to microwave irradiation. More energy is needed to achieve the same degree of solubilisation of anaerobic sludge compared to aerobic sludge. To assure minimal solubilisation, the maximal specific energy level to evaluate the effects on the performance is set at

of the anaerobic microbial community due to microwave irradiation. Finally, the SMA experiment revealed no significant differences in biogas and methane production due to the microwave treatment. Based on these results it can be concluded that low energetic microwave irradiation has no beneficial effects on the progress and effectiveness of the anaerobic digestion process, nor on the activity of the anaerobic microbial community.

Acknowledgements The authors would like to thank the Agency for Innovation by Science and Technology Flanders (IWT 121499) and the KU Leuven Research Council (OT/13/063) for the financial support.

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