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Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc
Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure Kaoutar Aboudi,* Carlos José Álvarez-Gallego, and Luis Isidoro Romero-García Department of Chemical Engineering and Food Technology, Faculty of Sciences, Agrifood Campus of International Excellence (CeiA3), University of Cádiz, 11510 Puerto Real, Cadiz, Spain Received 30 July 2015; accepted 5 October 2015 Available online xxx
The effect of mesophilic anaerobic co-digestion of dried pellets of exhausted sugar beet cossettes (ESBC-DP) and cow manure (CM) on the enhancement of methane generation and process stability were studied with the aim to select the best substrate mixture ratio. A series of batch experiments was conducted using the following five mixture ratios of ESBC-DP:CM: 0:100; 25:75; 50:50; 75:25 and 100:0. Best results were obtained from mixture ratios with ESBC-DP proportions in the range of 25e50%. Mixture ratio of 50:50 showed a specific methane production (SMP) increase of 81.4% and 25.5%, respectively, in comparison with mono-digestion of ESBC-DP and CM. Evolution of the indirect parameter named acidogenic substrate as carbon (ASC) could be used to provide more insight about the process stability of anaerobic digestion. ASC accumulation was observed in reactors with higher ESBC-DP proportions leading to a delay in VFAs consumption and conversion into methane. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Anaerobic co-digestion; Methane generation; Sugar beet by-product; Cow manure; Process stability; Acidogenic substrate as carbon]
In recent years, the expanding use of biomass as an energy source forms a major part of the global energy system by increasing its use as a feedstock for biofuel production and contributing in reducing carbon dioxide emissions and other pollutants for the global warming (1). Additionally, using biomass as a resourceful material for renewable energy production could prevent the landfilling of the wastes which creates environmental hazards (2). Agri-food industries generate large amounts of waste biomass, which can be used in waste-to-energy processes (3,4). At this juncture, anaerobic digestion (AD) proved to be a robust process to generate energy from organic waste, where the economy and the environment are in balance with each other (5). The high carbohydrate content in agri-food wastes is considered as the main component for biogas production by means of AD process. Lignocellulosic agri-food wastes, such as exhausted sugar beet cossettes (ESBC), can be used as a raw material for biogas production since they are composed mainly of carbohydrates, cellulose, hemicelluloses, and lignin material (6,7). ESBC are a by-product of the beet sugar industry, formed from the fibrous residues of sugar beets (Beta vulgaris) after several extraction processes (7). The dried pellets (DP) of ESBC used in this study are composed of 85% beet pulp and 15% molasses and have a high lignocellulosic-type organic matter (OM) content (90% volatile solids, VS) and a nitrogen deficiency (6,8). From other hand, the carbonaceous substrate ESBC has low nitrogen content which is a necessary nutrient for AD. Therefore, in
* Corresponding author. Tel.: þ34 956016474; fax: þ34 956016411. E-mail addresses: [email protected] (K. Aboudi), [email protected] (C.J. Álvarez-Gallego), [email protected] (L.I. Romero-García).
order to achieve efficient biogas production, supplementation of pH buffer and minerals is essential to optimize the pH conditions and nutrient balance (9,10). However, the cost of operation could be considerably increased. It has been reported that co-digestion can be an interesting option for improvement in biogas yields during AD of lignocellulosic wastes due to the positive synergisms established in the digestion process by providing a better nutritional balance (10e15). Livestock wastes such as animal manure (cow manure, CM, in present study) not only contribute nitrogen (nutrient balance) and alkalinity (buffering capacity) but also provide a high microbiological activity, which is able to degrade vegetal fiber as well as diluting the effect of toxic compounds contained in agri-food wastes (7,16,17). Some studies about AD of sugar beet by-product were found in literature (6e8,18e22), however, few of them were focused on the treatment of exhausted sugar beet pulp as dried pellets (6,8). The pellets of ESBC are usually used as animal feedstock which is not a compromising strategy for sugar beet plants processing due the high energetic cost of this industry. The use of sugar beet by-product for clean energy production such as biogas richmethane could offset the costs of production of sugar from sugar beet. Ohuchi et al. (18) studied the thermophilic anaerobic codigestion of sugar beet tops silage (SBT) with dairy manure (DM) at four SBT silage proportions. The highest methane yield of 422 mL/gVS and VS reduction of 57%, were obtained when the mixture contained the lowest SBT proportion (40%) while the system failure was observed for the highest SBT proportion. Similarly, Umetsu et al. (20) studied the thermophilic anaerobic co-digestion of sugar beet by-products (tops and roots) with DM in both batch and semi-continuous systems. For batch experiment, the authors
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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observed that methane production was stopped at 30% (tops and roots) and 15% (roots) beet by-products additions to DM, however, SBT proportions at 40% have shown the maximum methane yield. Subsequently, in semicontinuous operation, the authors observed that the mixture with 40% beet tops have shown the highest cumulative methane production increasing by around 60% over the digestion of DM as a sole substrate. Aboudi et al. (6) also investigated mesophilic AcoD of dried pellets of exhausted sugar beet cossettes (ESBC-DP) with pig manure (PM) as animal co-substrate. They reported that the PM addition in the proportion of 68% caused a significant increase in methane yields by creating a synergistic effect in the AD medium. In another attempt, Lehtomaki et al. (21) studied the mesophilic AcoD of SBT with different crop residues and CM. They analysed the effect of substrate proportions in the mixtures. They reported that at a crop proportion of 30% (corresponding to 15e19% of SBT), the specific methane production (SMP) increased 32% with respect to the digestion of CM as a sole substrate. So far no study has been conducted on co-digestion of ESBC-DP and CM. Therefore, the present study was carried out to evaluate the AcoD of ESBC-DP and CM at mesophilic conditions using five different mixture ratios of the substrates with the objective to find out the best combination between the selected substrates to achieve best methane production and process stability. MATERIALS AND METHODS Substrates and inoculum The ESBC as DP was obtained from a sugar beet processing plant at Jerez de la Frontera (Cádiz) in the south of Spain, during the summer harvesting period. Samples were stored at 4 C to avoid its degradation at room temperature. The used ESBC-DP had around 20e70 mm of length and 6 mm diameter and its TS content ranged 80e90%. The pellets of ESBC are composed of 85% of beet pulp and 15% of residual molasses. CM was collected from a semi-intensive farm facilities located at El Puerto de Santa Maria (Cádiz) in the south of Spain and was used the same day of its collection. The farm does not have any system for separation at source of urine and faeces. Mesophilic anaerobic effluent from a laboratory scale semi-continuous reactor fed with ESBC-DP was used as inoculum. The reactor was operated previously, at stable conditions, for about two years and the stabilised methane yield was 280 mLCH4/gVSadded. The physico-chemical characteristics of the substrates and inoculum are summarized in Table 1. Experimental start-up and operation Batch tests were conducted using a series of 2 L working volume stainless steel reactors (dimensions: 160 260 650 mm). The reactors have a glass cover with several ports including an inlet port for feeding and an output port for biogas collection. The temperature (35 0.5 C) was maintained during all the experiments by a heating plate located at the base of each reactor which was covered by a metal jacket for better heat transfer. Temperature was continuously measured by an inner temperature sensor and controlled by a proportional-integral-derivative (PID) control system. The mixing system consisted in an independent motor agitation and a stirring blade for each reactor and the stirring rate was maintained at 18 rpm (6). At the start of assays, reactors were inoculated at 50% (dry basis) with the mesophilic inoculum commented before. The following ESBC-DP:CM mixture ratios were tested: 0:100, 25:75, 50:50, 75:25 and 100:0. The total solids (TS) content of the mixtures was
TABLE 1. Physicochemical characteristics of substrates (ESBC-DP and CM) and inoculum. Component pH TS VS DOC sCOD tCOD TVFA Alkalinity N-NH4 TKN Ratio C/N a
a
Unit
ESBC-DP
CM
Inoculum
e g/kg (%TS) gC/kg gO2/kg gO2/kg gHAc/kg gCaCO3/kg gN/kg gN/kg (TS) e
5.840.12 874.50.12 88.90.35 37.840.15 480.26 120.720.24 5.80.12 3.30.16 0.180.14 15.41.02 33.50.2
6.170.2 221.30.28 77.80.24 12.930.42 15.10.15 86.250.32 2.6 0.48 34.60.25 2.481.38 36.10.63 12.50.16
7.320.14 36.90.18 20.80.15 3.840.18 9.680.22 15.380.22 1.335.2 42.226.8 0.290.52 5.70.54 21.20.26
ESBC en pellets form lixiviated sample with deionized water.
adjusted to 8% (8). The tests were carried out in duplicate. The initial pH was measured and was adjusted to the required pH (10) by adding an alkali (NaOH, 8M). Reactors were hermetically sealed and then were initially flushed with nitrogen gas to remove any residual oxygen. Tests were run until no further production of biogas was observed (approaching zero). Analytical methods To characterise the substrates and control the process, the following parameters were analysed: pH, total solids (TS), VS, total and soluble chemical oxygen demand (tCOD and sCOD), dissolved organic carbon (DOC), volatile fatty acids (VFAs), ammoniacal and total Kjeldahl nitrogen (NH4-N and TKN) and alkalinity. All analytical determinations were performed according to standard methods (23). The pH was measured directly from the samples using a CrisonBasic20 pH meter (Crison Instrument, Spain). For determinations of sCOD, DOC and VFAs, the samples were previously lixiviated with deionised water, during 2 h, and filtrated by 0.47 mm glass fiber filter according to Álvarez-Gallego et al. (24). Samples for VFAs measurement were filtrated again with 0.22 mm Teflon filter. The parameters tCOD, TS and VS determinations were performed directly without lixiviation. DOC analysis was carried out in an Analytic-Jena multi NC 3100 carbon analyser with chemiluminescence detector (CLD) by infraredcombustion method (5310B) of Standard Method using the oxygen 5.0 at pressure of 4e6 bars. The VFAs analysis was carried out using a gas chromatograph Shimadzu GC-2010 equipped with a flame ionisation detector (FID) and capillary column filled with Nukol. Hydrogen was used as carrier gas with a flow rate of 50 ml/min (6). In addition, N2 as make-up gas and synthetic air as comburent gas were used. Biogas was collected in a 10 L Tedlar gas bag (SKC, UK) and its volume was measured daily using a high precision wet drum-type gas meter (Ritter TG5). The gas composition was determined by using a gas chromatograph (Shimadzu GC-2014) with a stainless steel column packed with Carbosieve SII (diameter of 3.2 mm and 3.0 m length) and thermal conductivity detector (TCD). Helium was used as a carrier gas with flow rate of 30 ml/min. New indirect parameters for the AD performance interpretation In order to gain a better knowledge of the co-digestion of ESBC-DP with CM and its effect on the effectiveness of AD process, new indirect parameters were analysed, basing on the classical analytical determinations, which provide additional information about the process evolution. Fdez-Güelfo et al. (25) have established these new indirect parameters to evaluate the AD process performance and especially to understand the relationship between the different microbiological stages of the AD process and the effect on the system stability. According to the authors, the parameter dissolved acid carbon (DAC) represents the content in carbon associated to VFAs and it can be obtained from the VFA concentration in the medium (considering the relation between the carbon weight and the molecular weight in each VFA). Moreover, the acidogenic substrate as carbon (ASC) parameter is related to the soluble OM (expressed in carbon terms) which has been not transformed into VFAs. Thus, ASC can be obtained from the difference between the DOC and the DAC: ASC ¼ DOC DAC
(1)
The new indirect parameter, ASC, can be used as an indicator to study the linkage between each AD stage and the next ones; i.e., to indicate if the process is balanced and the stages are coupled. Thus, an increase of ASC can be produced when the hydrolysis rate is higher than acidogenesis rate, as it is observed usually at the beginning in a batch AD test. Besides, a decrease in ASC can be expected in the opposite case when most of the OM has been hydrolysed and its conversion into organic acids predominates. Furthermore, a supported increase in DAC can be associated to an imbalance between acidogenesis and methanogenesis, related to higher metabolic rate of acidogenic microorganisms. Finally, decreasing in DAC is normally related to methane production through methanogenic activity. In short, it can be pointed out than accumulation of ASC in the process can be related to problems in the acidogenic stage while DAC accumulations are related to problems in methanogenic stage.
RESULTS AND DISCUSSION Characterisation of ESBC-DP and CM The characteristics of the two substrates can be observed in Table 1. As can be seen, the used dried pellets of ESBC are a carbonaceous-type material with a high solid content (high VS and COD values). On the contrary, total nitrogen and alkalinity contents of CM are 2.3 and 10.5 times higher than for ESBC-DP. In consequence, the selected wastes are complementary and their mixture can lead to a suitable nutrient balance, offsetting the deficiencies of each one. In AcoD process, the ratio between the contents of carbon and nitrogen (C/N ratio) in the feedstock is considered as one of the critical parameters for process performance. The range of 20e30 for
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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16000 14000
VFAs(mgAcH/L)
12000 10000
TVFA
8000
HAc HPr
6000
HBu 4000
HVal
2000 0 (0:100)
(25:75)
(50:50)
(75:25)
(100:0)
(ESBC-DP:CM) mixture raƟos FIG. 1. Total volatile fatty acidity (TVFA) and maximum levels of predominant VFAs for the different mixture ratios tested in the co-digestion study.
the C/N ratio has been considered as the optimum in several literature papers (9,26). However, other authors have proposed lower ranges, indicating that optimum C/N ratio is depending on the waste characteristics (16,27). In the present work, mesophilic anaerobic co-digestion (AcoD) of ESBC-DP and CM was studied, testing the following five mixtures ESBC-DP:CM: 0:100, 25:75, 50:50, 75:25 and 100:0, corresponding to the C/N ratios of 12.5:1, 18.5:1, 23.5:1, 28.5:1 and 33.5:1, respectively. The ESBC-DP used in this work had a very high TS content (874.5 g/kg) and were composed mainly by beet pulp (85%) and molasses (15%). It should be noted that few papers on AD treatment applied to this by-product of the sugar beet industries have been found in literature (6,8). Analysis of the system stability through acidity related parameters Different types of parameters can be used to evaluate the AD process stability. According to literature, pH is one of the most critical variables for biomethanisation process stability. However, the pH value is the result of the acid-base equilibria in the system and, hence, it involves the VFAs productions and the substrates alkalinity. Therefore, in this section, the more important parameters related to the maintaining of pH in the medium have been grouped: pH, total volatile fatty acidity (TVFA), and the two ratios acidity/alkalinity and propionic to acetic acids (HPr/HAc). The pH affects significantly the growth rate of the microorganisms capable to transform the OM into biogas (28). For biomethanisation processes, pH values should be in the range of 6.8e8.5 (10,29). In the present work, during the first days of the process operation, pH dropped below the required range, especially for the reactors with high ESBC-DP content. It can be mainly due to the low alkalinity of ESBC-DP and to the VFAs liberation in the medium (Fig. 1.). As a consequence, pH control with alkali (NaOH, 8M) was necessary once a day during the start-up period, for all
3
reactors, to avoid initial system acidification. However, it was observed that for reactors with mixture ratios of 0:100, 25:75 and 50:50, the pH was stabilized promptly and it remained in the appropriate range, without NaOH addition, after 5, 7 and 8 days, respectively. The short period necessary to reach the suitable pH for the AD process in those reactors with a high CM proportion was mainly related to the high buffer capacity supplied by the livestock substrate (CM) and also the microorganisms variety coming from the ruminant animal tract which are adapted to degrade vegetal fibers (16,17). In Table 2, the initial and final pH values for the different mixture ratios of ESBC-DP and CM are shown. At the end of the process, pH of all reactors was in the required range. Fig. 1 shows the maximum TVFA concentration and levels of the predominant VEAs produced during the assay for the different mixture ratios tested. Acetic acid (HAc) and propionic acid (HPr) were the main VFAs produced along the tests for all mixture ratio reactors. Furthermore, the results point out that the TVFA content increases with increase in the ESBC-DP proportion in the mixtures. Likewise, the maxima for individual VFAs, and especially the maxima propionic acid concentrations, were reached for mixtures with higher ESBC-DP contents. For the reactors 75:25 and 100:0, TVFA started decreasing only after 20 days and 30 days of the assay, respectively. This issue was likely related to the lower buffer capacity of those reactors compared with reactors with high content of CM. As mentioned previously from the characteristics of the substrates, CM has higher nitrogen content and alkalinity. Synergistic effect between carbonaceous wastes and livestock manures was reported to be the reason of neutralizing the VFAs generated, and hence a well operation of the digester (11). The inhibition effect of the propionic acid on AD process is well known (30). Accordingly, acidification was observed in the reactor with the mixture ratio (100:0) producing a delay in biogas production as observed in Fig. 3. However, and despite of the high VFAs accumulation, after a long period of adaptation of the microorganisms, TVFA decreased and biogas was produced. This fact was probably due to the use of an inoculum especially well acclimatized to ESBC-DP treatment as it is coming from a semicontinuous reactor fed with ESBC-DP. On the contrary, the CM addition enhanced the buffer capacity, neutralising the VFAs production and avoiding the acidification and the failure of the system (31,32). Hence, no acidification was produced for reactors with CM proportions above 50%. The CM addition has permitted to maintain an optimal pH for methanogens and to balance the nutrients content in the medium. One of the main criteria to predict the system failure is established by the ratio acidity/alkalinity which permits to analyse the process stability and the balance between liberation of organic acids and the buffer capacity in AD reactors. Fig. 2A shows that for the reactors 0:100 and 25:75 the acidity/alkalinity ratio was in the optimum range, while for reactor 50:50 was slightly higher than 0.4 although limit values for system failure were not exceeded (33). However, when high proportions of ESBC-DP were added to the mixtures, and especially for reactor 100:0, the acidity/alkalinity
TABLE 2. Summarized comparison of the co-digestion process performance for the tested mixture ratios. Mixture ratios (ESBC-DPa:CM)
C/N ratio
SMP (mLCH4/gVSadded)
DSMP /(100:0)
DSMP /(0:100)
(%)
(%)
Methane production rate (LCH4/Lreactor d)
(0:100) (25:75) (50:50) (75:25) (100:0)
12.5 18.5 23.5 28.5 33.5
447.121.8 557.412.2 560.16.4 323.70.9 308.815.6
44.828.7 80.517.1 81.411.5 4.812.7 e
e 24.723.4 25.316.8 27.68.3 30.917.5
0.650.08 1.220.11 0.940.18 0.700.10 0.540.27
a
Removal efficiency (%)
pH
tCOD
VS
Initial pH
Final pH
64.63.5 65.87.2 54.211.8 57.33.8 51.411.2
58.22.4 77.51.6 72.11.8 65.32.1 60.52.4
6.80.1 6.90.1 70.1 70.2 7.10.1
8.30.1 8.20.1 8.30.1 8.20.1 8.20.1
ESBC-DP was composed of 85% of pulp and 15% of molasses.
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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(A)
(0:100)
J. BIOSCI. BIOENG.,
(25:75)
(50:50)
(75:25)
(100:0)
acidity/alkalinity raƟo
1.6
1.2
0.8
0.4
0 0
20
40
60
Time (Days)
(B)
(0:100)
(25:75)
(50:50)
(75:25)
(100:0)
10
HPr/HAc raƟo
8
6
4
2
0 0
20
Time (days)
40
60
FIG. 2. Evolution of the ratios acidity/alkalinity (a) and HPr/HAc (b) for the different mixture ratios tested in the co-digestion study.
ratio was over limit values for a long period of the assay before admissible values were reached at the end of the test. Moreover, another widely used parameter to analyse process stability is the ratio HPr/HAc (34e36). In literature, it has been reported that the maximum value for the HPr/HAc ratio is 1.4 and destabilization occurs when this value is exceeded (37). However, other authors have proved that this limit value can vary depending on the characteristics and nature of the substrates and reactor performance (36). Furthermore, Pullammanappallil et al. (34) deduced that conditions that cause inhibition in one system do not necessarily affect to all AD systems. As can be seen in Fig. 2B, in the present work, the HPr/HAc ratio was maintained in the range 0e2 for the reactors 0:100 and 25:75 throughout the test. However, relatively high values were obtained for the reactors 50:50 and 75:25, especially between days 32e42 and 37e48, respectively. Further, for the reactor 100:0, a sharp increase in the HPr/HAc ratio was observed around day 55, reaching a maximum value of 10. Despite of the high values of the HPr/HAc ratio reached in reactors with higher EBSC-DP content (50:50; 75:25 and 100:0), the reactors were able to remove progressively HPr and the ratio started decreasing continuously to achieve more admissible values. Thus, the higher HPr/HAc ratios could be associated to longer periods to reach the VFAs removal and higher lagphase for biogas production. This behaviour can be interpreted by the destabilisation of these reactors due to the uncoupling between metabolic rates of the two involved microorganism groups, the acid producers and the consumers. According to obtained results in the present study, the HPr/HAc ratio of 2 can be considered as an admissible value for a proper
performance of the AD system for ESBC-DP and CM co-digestion at mesophilic conditions. In literature, the specific concentration of HPr leading to AD inhibition is not clear. In fact, a wide variation in HPr concentrations has been considered as inhibitory for the AD process. Thus, Barredo and Evison (38) have considered 1500e2220 mg/L of HPr as the limit range for the AD failure. On the contrary, McCarty and Brosseau (39) have reported that HPr concentrations up to 8000 mg/L can be tolerated by anaerobic digesters if the system is working correctly. The same conclusion was reached by Pullammanappallil et al. (34). In the present study, the maximum HPr concentrations were in the range of 1099e5500 mg/ L and the system was not inhibited since it was able to degrade the accumulated HPr and convert it into biogas as commented previously. Maximum propionic acid concentrations of 1098.8 mg/L, 1935.7 mg/L, 3126.5 mg/L, 35,556.4 mg/L and 5438.36 mg/L were detected for reactors 0:100, 25:75, 50:50, 75:25 and 100:0, respectively. It can be pointed out that propionic acid degradation is a specific activity of acetogenic microorganism populations and its acclimatisation to high HPr concentrations is essential for develop it. For hence, the utilisation in this study of an inoculum well adapted to high HPr concentrations produced by ESBC-DP degradation has been critical. Analysis of the process stability based on the indirect carbon-related parameters As previously commented in the M&M section, and for a better understanding of the relationship between organic acids accumulation, biogas production and the proportions of ESBC dried pellets in the feed of the reactors, a series of parameters have been determined based on those developed by Fdez-Güelfo et al. (25). Fig. 3 shows the evolution of the following parameters: DOC, DAC, ASC and SMP for the different mixture ratios tested. As can be observed in Fig. 3, an initial increase for the new parameter ASC was observed in all reactors, as well as for DOC. These evolutions were due to the hydrolysis of the particulate OM of the wastes, but the ASC increase indicated that transformation in VFAs was not completely coupled initially with hydrolysis rate. The ASC increase was much more pronounced for those mixture ratios with ESBC-DP proportions above 50% (Fig. 3D and E). Additionally, it is clear from Fig. 3 that both the time required to reach the maximum value of ASC as the value of these maxima increased with increasing ESBC-DP content in the mixtures. Thus, time periods of 6, 4, 12, 25 and 26 days were necessary to reach the ASC maxima of 2212.8, 2438.2, 4099.1, 6662.6 and 7367.8 mgC/L for the mixture ratios 0:100, 25:75, 50:50, 75:25 and 100:0 respectively. This fact, revealed that the hydrolysis rate was higher or equal than the acidogenesis rate during the above mentioned periods, leading to the ASC accumulation observed in each case. Similar behavior was reported by Romero-Aguilar et al. (40) when studying AD of the organic fraction of municipal solid wastes for biohydrogen production. Authors reported that when ASC values were higher than DOC, the acidogenic stage was limited or inhibited. However, equilibrium between ASC and the DOC indicates a well coupling of hydrolysis and acidogenesis phases. Subsequently, when the hydrolysis rate decreased, the ASC values dropped quickly. In this case a minor quantity of OM was available to be hydrolysed and solubilized in the medium and the acidogenesis rate was higher than the hydrolysis rate. The ASC decrease was related to its consumption and transformation into VFAs by acidogenic microorganisms. However, the decreasing in ASC was not accompanied with an increase in DAC but rather with the increase in methane production. This fact is indicative that acidogenic and methanogenic activities were coupled and well balanced and VFAs were transformed into methane as soon as they were formed. Only a slight deviation
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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FIG. 3. Specific methane production (expressed as mLCH4/gVSadded), dissolved organic carbon (DOC), dissolved acid carbon (DAC) and acidogenic substrate as carbon (ASC) for the different mixture ratios tested in the co-digestion study.
between times required to reach the maxima of ASC and DAC was observed for the reactor 100:0. Moreover, in this case, biogas production started once DAC decreased. In short, the evolution of the reactors is highly related with the ESBC-DP proportion in the mixture. Thus, the reactors with minor contents in ESBC-DP (the mixtures 0:100 and 25:75) showed a similar behaviour in the ASC trend, and the different stages of the process e hydrolysis, acidogenesis and methanogenesis e were well coupled. On the contrary, the reactors with higher contents in ESBC-DP (the mixtures 75:25 and 100:0) have also shown a similar evolution. In these cases, decoupling was observed between hydrolytic and acidogenic stages, leading to ASC accumulations.
However, inhibition was not observed for the methanogenic stage. The reactor 50:50 shows an intermediate comportment. Enhancement in the AcoD efficiency: OM removal and methane production The main parameters to analyse the performance of the mesophilic batch anaerobic digesters used in this study are shown in Table 2. According to the obtained results, the higher tCOD removal was obtained for the reactors 0:100 and 25:75 reaching values above 64%. For the rest of reactors, tCOD removal ranged of 51e58%. Regarding the effect of the co-digestion on methane production, the obtained results showed that all the tested mixture ratios were
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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better than the mono-digestion of ESBC-DP. Table 2 shows that increments of specific methane production (SMP, expressed as mLCH4/ gVSadded) with respect to the mixture ratio 100:0 were of 80.5% and 81.4% for the mixtures 25:75 and 50:50 respectively, while only a slight increase (4.8%) was obtained for the mixture 75:25. Methane production rates (MPR, expressed as LCH4/Lreactor d) are shown in Table 2. As can be observed, the highest MPRs were observed for reactors 25:75 and 50:50 of about 1.22 0.11 LCH4/ Lreactor d and 0.94 0.18 LCH4/Lreactor d, respectively. Individual digestion of CM and ESBC-DP showed lower MPRs of 0.65 0.08 LCH4/Lreactor d and 0.54 0.27 LCH4/Lreactor d, respectively. Furthermore, analysing the increments of SMP with respect to the mixture 0:100, which corresponds to the digestion of CM as a sole substrate, results showed that the mixtures 25:75 and 50:50 improved the SMP in a 24.7% and 25.3%, respectively. In short, the addition of ESBC-DP to CM above 50% affected negatively the methane production with respect to anaerobic mono-digestion of CM. Another important issue that supports the feasibility and the improvement due to the ESBC-DP co-digestion with CM is related to the required time for process start-up. From the results of the SMP in Fig. 3, it can be concluded that the lag-phase period for methane production can be reduced significantly when the ESBC-DP was codigested with CM. Besides, the lag-phase period was lower for the greater proportion of CM in the mixture. The minimum lag-phase period of 6 days was observed for the mixture 25:75, instead of 37 days for the digestion of ESBC-DP individually or 9 days for the CM mono-digestion. The lag-phase is the time required by microorganisms for their adaptation to the environment conditions. Thus, considering the complex nature of ESBC-DP (lignocellulosic-type substrate), it can be expected that the microorganisms needed a longer adaptationtime for mixtures with higher ESBC-DP proportions. In this work it has been observed that lag-phase was shortened by CM addition, likely due to that livestock wastes provided microorganisms and all the macro and micro nutrients required for the microbial growth from the rumen (16,17), creating a synergistic effect in the medium (11). Moreover, the big lag-phase periods observed for the reactors with ESBC-DP proportions above 50% were also related to the higher levels of the ASC accumulated in the medium in these tests. Hence, the delay in methane production was associated to the transformation of ASC into organic acids (delay in acidogenesis stage) because no VFAs accumulation was observed. Once the systems were adapted to the high ASC concentrations, the organic acid production started at the same time as its transformation into methane as discussed in the previous section. This behaviour indicates a well coupling of the acidogenic and methanogenic phases. A comparison with other works focusing on co-digestion of similar substrates was reported in a previous paper (6), and recently published works were also compared to the present research. As a conclusion, it can be pointed out that the results obtained in the present study about individual digestion of ESBCDP were in the inferior limit of those obtained by other authors (7), and quite comparable to several studies (6,19). However, the optimal result obtained for co-digestion process in the current research were in the superior limit of other works. Fang et al. (7), obtained SMPs of 490, 500 and 240 mlCH4/gVSadded, respectively for AD of sugar beet leaves, tops and sugar beet pulp. Similarly, Gissén et al. (41) studied AD of sugar beet by-product and obtained a SMP of 419 mlCH4/gVSadded. The investigation performed by Montañes et al. (22) using sugar beet pulp lixiviation and sewage sludge showed a SMP of 544 mlCH4/gVSadded. Aboudi et al. (6) obtained a SMP of 494 mlCH4/gVSadded from AcoD of ESBC with PM. In another attempt, Aboulenein et al. (42) obtained very high SMP of 695 mlCH4/gVSadded when studying AcoD of CM with agricultural wastes (coconut, cassava pulp, coffee grounds).
J. BIOSCI. BIOENG., According to the discussed parameters trends, the mixtures 25:75 and 50:50 had been the best mixtures tested in this study with high methane production and process stability while ESBC-DP addition above 50% affected negatively the AcoD process. ACKNOWLEDGMENTS This work was supported by the Spanish Ministry of Economy and Finance-ERDF for the IþDþI project (CTM2013-43938-R) and co-funded by the MICINN-Spain (UNCA08-1E-035 Project). The authors acknowledge to the University of Cadiz (Spain) for the Scholarship UCA-2010-063PU/EPIF-FPI-A/BC and the Agrifood Campus of International Excellence (Ceia3). The authors also wish to thank the Spanish Ministry of Science and Innovation (PROBIOGAS Project PS-120000-2007-6). References 1. Sawatdeenarunat, C., Surendra, K. C., Takara, D., Oechsner, H., and Khanal, S. K.: Anaerobic digestion of lignocellulosic biomass: challenges and opportunities, Bioresour. Technol., 178, 178e186 (2015). 2. Weiland, P.: Biogas production: current state and perspectives, Appl. Microbiol. Biotechnol., 85, 849e860 (2010). 3. Ward, A. J., Hobbs, P. J., Holliman, P. J., and Jones, D. L.: Optimisation of the anaerobic digestion of agricultural resources, Bioresour. Technol., 99, 7928e7940 (2008). 4. Anwar, Z., Gulfraz, M., and Irshad, M.: Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review, J. Radiat. Res. Appl. Sci., 7, 163e173 (2014). 5. Nishio, N. and Nakashimada, Y.: Recent development of anaerobic digestion processes for energy recovery from wastes, J. Biosci. Bioeng., 103, 105e112 (2007). 6. Aboudi, K., Álvarez-Gallego, C. J., and Romero-García, L. I.: Improvement of exhausted sugar beet cossettes anaerobic digestion process by co-digestion with pig manure, Energy Fuels, 29, 754e762 (2015). 7. Fang, C., Boe, K., and Angelidaki, I.: Anaerobic co-digestion of by-products from sugar production with cow manure, Water Res., 45, 3473e3480 (2011). 8. Hutnan, M., Drtil, M., and Mrafkova, L.: Anaerobic biodegradation of sugar beet pulp, Biodegradation, 11, 203e211 (2000). 9. Wang, X., Yang, G., Feng, Y., Ren, G., and Han, X.: Optimizing feeding composition and carbon-nitrogen ratios for improved methane yield during anaerobic co-digestion of dairy, chicken manure and wheat straw, Bioresour. Technol., 120, 78e83 (2012). 10. Zoetemeyer, R. J., van den Heuvel, J. C., and Cohen, A.: pH influence on acidogenic dissimilation of glucose in an anaerobic digestor, Water Res., 16, 303e311 (1982). 11. Pagés-Díaz, J., Pereda-Reyes, I., Taherzadeh, M. J., Sárvári-Horváth, I., and Lundin, M.: Anaerobic co-digestion of solid slaughterhouse wastes with agroresidues: synergistic and antagonistic interactions determined in batch digestion assays, Chem. Eng. J., 245, 89e98 (2014). 12. Mata-Alvarez, J., Macé, S., and Llabrés, P.: Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives, Bioresour. Technol., 74, 3e16 (2000). 13. Cuetos, M. J., Fernández, C., Gómez, X., and Morán, A.: Anaerobic co-digestion of swine manure with energy crop residues, Biotechnol. Bioprocess Eng., 16, 1044e1052 (2011). 14. Hartmann, H. and Ahring, B. K.: Anaerobic digestion of the organic fraction of municipal solid waste: influence of co-digestion with manure, Water Res., 39, 1543e1552 (2005). 15. Tian, H., Duan, N., Lin, C., Li, X., and Zhong, M.: Anaerobic co-digestion of kitchen waste and pig manure with different mixing ratios, J. Biosci. Bioeng., 120, 51e57 (2015). 16. Kivaisi, A. K. and Eliapenda, S.: Application of rumen microorganisms for enhanced anaerobic degradation of bagasse and maize bran, Biomass Bioenergy, 8, 45e50 (1995). 17. Hindrichsen, I. K., Wettstein, H. R., Machmüller, A., Knudsen, K. E. B., Madsen, J., and Kreuzer, M.: Digestive and metabolic utilisation of dairy cows supplemented with concentrates characterised by different carbohydrates, Anim. Feed Sci. Technol., 126, 43e61 (2006). 18. Ohuchi, Y., Ying, C., Lateef, S. A., Ihara, I., Iwasaki, M., Inoue, R., and Umetsu, K.: Anaerobic co-digestion of sugar beet tops silage and dairy cow manure under thermophilic condition, J. Mater. Cycles Waste Manage., 17, 540e546 (2014). 19. Alkaya, E. and Demirer, G. N.: Anaerobic mesophilic co-digestion of sugar-beet processing wastewater and beet-pulp in batch reactors, Renew. Energy, 36, 971e975 (2011).
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20. Umetsu, K., Yamazaki, S., Kishimoto, T., Takahashi, J., Shibata, Y., Zhang, C., Misaki, T., Hamamoto, O., Ihara, I., and Komiyama, M.: Anaerobic codigestion of dairy manure and sugar beets, Int. Congr. Ser., 1293, 307e310 (2006). 21. Lehtomäki, A., Huttunen, S., and Rintala, J. A.: Laboratory investigations on co-digestion of energy crops and crop residues with cow manure for methane production: effect of crop to manure ratio, Resour. Conserv. Recycl., 51, 591e609 (2007). 22. Montañés, R., Solera, R., and Pérez, M.: Anaerobic co-digestion of sewage sludge and sugar beet pulp lixiviation in batch reactors: effect of temperature, Bioresour. Technol., 180, 177e184 (2015). 23. American Public Health Association: Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, DC (2005). 24. Álvarez-Gallego, C.: Testing different procedures for the start up of a dry anaerobic co-digestion process of OFMSW and sewage sludge at thermophilic range, PhD thesis. University of Cádiz, Spain (2005). 25. Fdez-Güelfo, L. A., Álvarez-Gallego, C., Sales, D., and Romero, L. I.: New indirect parameters for interpreting a destabilization episode in an anaerobic reactor, Chem. Eng. J., 180, 32e38 (2012). 26. Li, Y., Park, S. Y., and Zhu, J.: Solid-state anaerobic digestion for methane production from organic waste, Renew. Sust. Energy Rev., 15, 821e826 (2011). 27. Zhang, T., Liu, L., Song, Z., Ren, G., Feng, Y., Han, X., and Yang, G.: Biogas production by co-digestion of goat manure with three crop residues, PLoS One, 8, e66845 (2013). 28. Kumar, G., Bakonyi, P., Sivagurunathan, P., Nemestóthy, N., Bélafi-Bakó, K., and Lin, C.-Y.: Improved microbial conversion of de-oiled Jatropha waste into biohydrogen via inoculum pretreatment: process optimization by experimental design approach, Biofuel Res. J., 2, 209e2014 (2015). 29. Nyns, E. J., Demuynch, M., and Naveau, H.: Biomethanation, the paradox of a mature technology, energy from biomass, pp. 146e150, in: Proceedings of the 3rd International Conference on Biomass, Venice, Italy (1985). 30. Madsen, M., Holm-Nielsen, J. B., and Esbensen, K. H.: Monitoring of anaerobic digestion processes: a review perspective, Renew. Sustain. Energy Rev., 15, 3141e3155 (2011). 31. Zhen, G., Lu, X., Kobayashi, T., Li, Y.-Y., Xu, K., and Zhao, Y.: Mesophilic anaerobic co-digestion of waste activated sludge and Egeria densa:
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Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure Kaoutar Aboudi,* Carlos José Álvarez-Gallego, and Luis Isidoro Romero-García Department of Chemical Engineering and Food Technology, Faculty of Sciences, Agrifood Campus of International Excellence (CeiA3), University of Cádiz, 11510 Puerto Real, Cadiz, Spain Received 30 July 2015; accepted 5 October 2015 Available online xxx
The effect of mesophilic anaerobic co-digestion of dried pellets of exhausted sugar beet cossettes (ESBC-DP) and cow manure (CM) on the enhancement of methane generation and process stability were studied with the aim to select the best substrate mixture ratio. A series of batch experiments was conducted using the following five mixture ratios of ESBC-DP:CM: 0:100; 25:75; 50:50; 75:25 and 100:0. Best results were obtained from mixture ratios with ESBC-DP proportions in the range of 25e50%. Mixture ratio of 50:50 showed a specific methane production (SMP) increase of 81.4% and 25.5%, respectively, in comparison with mono-digestion of ESBC-DP and CM. Evolution of the indirect parameter named acidogenic substrate as carbon (ASC) could be used to provide more insight about the process stability of anaerobic digestion. ASC accumulation was observed in reactors with higher ESBC-DP proportions leading to a delay in VFAs consumption and conversion into methane. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Anaerobic co-digestion; Methane generation; Sugar beet by-product; Cow manure; Process stability; Acidogenic substrate as carbon]
In recent years, the expanding use of biomass as an energy source forms a major part of the global energy system by increasing its use as a feedstock for biofuel production and contributing in reducing carbon dioxide emissions and other pollutants for the global warming (1). Additionally, using biomass as a resourceful material for renewable energy production could prevent the landfilling of the wastes which creates environmental hazards (2). Agri-food industries generate large amounts of waste biomass, which can be used in waste-to-energy processes (3,4). At this juncture, anaerobic digestion (AD) proved to be a robust process to generate energy from organic waste, where the economy and the environment are in balance with each other (5). The high carbohydrate content in agri-food wastes is considered as the main component for biogas production by means of AD process. Lignocellulosic agri-food wastes, such as exhausted sugar beet cossettes (ESBC), can be used as a raw material for biogas production since they are composed mainly of carbohydrates, cellulose, hemicelluloses, and lignin material (6,7). ESBC are a by-product of the beet sugar industry, formed from the fibrous residues of sugar beets (Beta vulgaris) after several extraction processes (7). The dried pellets (DP) of ESBC used in this study are composed of 85% beet pulp and 15% molasses and have a high lignocellulosic-type organic matter (OM) content (90% volatile solids, VS) and a nitrogen deficiency (6,8). From other hand, the carbonaceous substrate ESBC has low nitrogen content which is a necessary nutrient for AD. Therefore, in
* Corresponding author. Tel.: þ34 956016474; fax: þ34 956016411. E-mail addresses: [email protected] (K. Aboudi), [email protected] (C.J. Álvarez-Gallego), [email protected] (L.I. Romero-García).
order to achieve efficient biogas production, supplementation of pH buffer and minerals is essential to optimize the pH conditions and nutrient balance (9,10). However, the cost of operation could be considerably increased. It has been reported that co-digestion can be an interesting option for improvement in biogas yields during AD of lignocellulosic wastes due to the positive synergisms established in the digestion process by providing a better nutritional balance (10e15). Livestock wastes such as animal manure (cow manure, CM, in present study) not only contribute nitrogen (nutrient balance) and alkalinity (buffering capacity) but also provide a high microbiological activity, which is able to degrade vegetal fiber as well as diluting the effect of toxic compounds contained in agri-food wastes (7,16,17). Some studies about AD of sugar beet by-product were found in literature (6e8,18e22), however, few of them were focused on the treatment of exhausted sugar beet pulp as dried pellets (6,8). The pellets of ESBC are usually used as animal feedstock which is not a compromising strategy for sugar beet plants processing due the high energetic cost of this industry. The use of sugar beet by-product for clean energy production such as biogas richmethane could offset the costs of production of sugar from sugar beet. Ohuchi et al. (18) studied the thermophilic anaerobic codigestion of sugar beet tops silage (SBT) with dairy manure (DM) at four SBT silage proportions. The highest methane yield of 422 mL/gVS and VS reduction of 57%, were obtained when the mixture contained the lowest SBT proportion (40%) while the system failure was observed for the highest SBT proportion. Similarly, Umetsu et al. (20) studied the thermophilic anaerobic co-digestion of sugar beet by-products (tops and roots) with DM in both batch and semi-continuous systems. For batch experiment, the authors
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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observed that methane production was stopped at 30% (tops and roots) and 15% (roots) beet by-products additions to DM, however, SBT proportions at 40% have shown the maximum methane yield. Subsequently, in semicontinuous operation, the authors observed that the mixture with 40% beet tops have shown the highest cumulative methane production increasing by around 60% over the digestion of DM as a sole substrate. Aboudi et al. (6) also investigated mesophilic AcoD of dried pellets of exhausted sugar beet cossettes (ESBC-DP) with pig manure (PM) as animal co-substrate. They reported that the PM addition in the proportion of 68% caused a significant increase in methane yields by creating a synergistic effect in the AD medium. In another attempt, Lehtomaki et al. (21) studied the mesophilic AcoD of SBT with different crop residues and CM. They analysed the effect of substrate proportions in the mixtures. They reported that at a crop proportion of 30% (corresponding to 15e19% of SBT), the specific methane production (SMP) increased 32% with respect to the digestion of CM as a sole substrate. So far no study has been conducted on co-digestion of ESBC-DP and CM. Therefore, the present study was carried out to evaluate the AcoD of ESBC-DP and CM at mesophilic conditions using five different mixture ratios of the substrates with the objective to find out the best combination between the selected substrates to achieve best methane production and process stability. MATERIALS AND METHODS Substrates and inoculum The ESBC as DP was obtained from a sugar beet processing plant at Jerez de la Frontera (Cádiz) in the south of Spain, during the summer harvesting period. Samples were stored at 4 C to avoid its degradation at room temperature. The used ESBC-DP had around 20e70 mm of length and 6 mm diameter and its TS content ranged 80e90%. The pellets of ESBC are composed of 85% of beet pulp and 15% of residual molasses. CM was collected from a semi-intensive farm facilities located at El Puerto de Santa Maria (Cádiz) in the south of Spain and was used the same day of its collection. The farm does not have any system for separation at source of urine and faeces. Mesophilic anaerobic effluent from a laboratory scale semi-continuous reactor fed with ESBC-DP was used as inoculum. The reactor was operated previously, at stable conditions, for about two years and the stabilised methane yield was 280 mLCH4/gVSadded. The physico-chemical characteristics of the substrates and inoculum are summarized in Table 1. Experimental start-up and operation Batch tests were conducted using a series of 2 L working volume stainless steel reactors (dimensions: 160 260 650 mm). The reactors have a glass cover with several ports including an inlet port for feeding and an output port for biogas collection. The temperature (35 0.5 C) was maintained during all the experiments by a heating plate located at the base of each reactor which was covered by a metal jacket for better heat transfer. Temperature was continuously measured by an inner temperature sensor and controlled by a proportional-integral-derivative (PID) control system. The mixing system consisted in an independent motor agitation and a stirring blade for each reactor and the stirring rate was maintained at 18 rpm (6). At the start of assays, reactors were inoculated at 50% (dry basis) with the mesophilic inoculum commented before. The following ESBC-DP:CM mixture ratios were tested: 0:100, 25:75, 50:50, 75:25 and 100:0. The total solids (TS) content of the mixtures was
TABLE 1. Physicochemical characteristics of substrates (ESBC-DP and CM) and inoculum. Component pH TS VS DOC sCOD tCOD TVFA Alkalinity N-NH4 TKN Ratio C/N a
a
Unit
ESBC-DP
CM
Inoculum
e g/kg (%TS) gC/kg gO2/kg gO2/kg gHAc/kg gCaCO3/kg gN/kg gN/kg (TS) e
5.840.12 874.50.12 88.90.35 37.840.15 480.26 120.720.24 5.80.12 3.30.16 0.180.14 15.41.02 33.50.2
6.170.2 221.30.28 77.80.24 12.930.42 15.10.15 86.250.32 2.6 0.48 34.60.25 2.481.38 36.10.63 12.50.16
7.320.14 36.90.18 20.80.15 3.840.18 9.680.22 15.380.22 1.335.2 42.226.8 0.290.52 5.70.54 21.20.26
ESBC en pellets form lixiviated sample with deionized water.
adjusted to 8% (8). The tests were carried out in duplicate. The initial pH was measured and was adjusted to the required pH (10) by adding an alkali (NaOH, 8M). Reactors were hermetically sealed and then were initially flushed with nitrogen gas to remove any residual oxygen. Tests were run until no further production of biogas was observed (approaching zero). Analytical methods To characterise the substrates and control the process, the following parameters were analysed: pH, total solids (TS), VS, total and soluble chemical oxygen demand (tCOD and sCOD), dissolved organic carbon (DOC), volatile fatty acids (VFAs), ammoniacal and total Kjeldahl nitrogen (NH4-N and TKN) and alkalinity. All analytical determinations were performed according to standard methods (23). The pH was measured directly from the samples using a CrisonBasic20 pH meter (Crison Instrument, Spain). For determinations of sCOD, DOC and VFAs, the samples were previously lixiviated with deionised water, during 2 h, and filtrated by 0.47 mm glass fiber filter according to Álvarez-Gallego et al. (24). Samples for VFAs measurement were filtrated again with 0.22 mm Teflon filter. The parameters tCOD, TS and VS determinations were performed directly without lixiviation. DOC analysis was carried out in an Analytic-Jena multi NC 3100 carbon analyser with chemiluminescence detector (CLD) by infraredcombustion method (5310B) of Standard Method using the oxygen 5.0 at pressure of 4e6 bars. The VFAs analysis was carried out using a gas chromatograph Shimadzu GC-2010 equipped with a flame ionisation detector (FID) and capillary column filled with Nukol. Hydrogen was used as carrier gas with a flow rate of 50 ml/min (6). In addition, N2 as make-up gas and synthetic air as comburent gas were used. Biogas was collected in a 10 L Tedlar gas bag (SKC, UK) and its volume was measured daily using a high precision wet drum-type gas meter (Ritter TG5). The gas composition was determined by using a gas chromatograph (Shimadzu GC-2014) with a stainless steel column packed with Carbosieve SII (diameter of 3.2 mm and 3.0 m length) and thermal conductivity detector (TCD). Helium was used as a carrier gas with flow rate of 30 ml/min. New indirect parameters for the AD performance interpretation In order to gain a better knowledge of the co-digestion of ESBC-DP with CM and its effect on the effectiveness of AD process, new indirect parameters were analysed, basing on the classical analytical determinations, which provide additional information about the process evolution. Fdez-Güelfo et al. (25) have established these new indirect parameters to evaluate the AD process performance and especially to understand the relationship between the different microbiological stages of the AD process and the effect on the system stability. According to the authors, the parameter dissolved acid carbon (DAC) represents the content in carbon associated to VFAs and it can be obtained from the VFA concentration in the medium (considering the relation between the carbon weight and the molecular weight in each VFA). Moreover, the acidogenic substrate as carbon (ASC) parameter is related to the soluble OM (expressed in carbon terms) which has been not transformed into VFAs. Thus, ASC can be obtained from the difference between the DOC and the DAC: ASC ¼ DOC DAC
(1)
The new indirect parameter, ASC, can be used as an indicator to study the linkage between each AD stage and the next ones; i.e., to indicate if the process is balanced and the stages are coupled. Thus, an increase of ASC can be produced when the hydrolysis rate is higher than acidogenesis rate, as it is observed usually at the beginning in a batch AD test. Besides, a decrease in ASC can be expected in the opposite case when most of the OM has been hydrolysed and its conversion into organic acids predominates. Furthermore, a supported increase in DAC can be associated to an imbalance between acidogenesis and methanogenesis, related to higher metabolic rate of acidogenic microorganisms. Finally, decreasing in DAC is normally related to methane production through methanogenic activity. In short, it can be pointed out than accumulation of ASC in the process can be related to problems in the acidogenic stage while DAC accumulations are related to problems in methanogenic stage.
RESULTS AND DISCUSSION Characterisation of ESBC-DP and CM The characteristics of the two substrates can be observed in Table 1. As can be seen, the used dried pellets of ESBC are a carbonaceous-type material with a high solid content (high VS and COD values). On the contrary, total nitrogen and alkalinity contents of CM are 2.3 and 10.5 times higher than for ESBC-DP. In consequence, the selected wastes are complementary and their mixture can lead to a suitable nutrient balance, offsetting the deficiencies of each one. In AcoD process, the ratio between the contents of carbon and nitrogen (C/N ratio) in the feedstock is considered as one of the critical parameters for process performance. The range of 20e30 for
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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16000 14000
VFAs(mgAcH/L)
12000 10000
TVFA
8000
HAc HPr
6000
HBu 4000
HVal
2000 0 (0:100)
(25:75)
(50:50)
(75:25)
(100:0)
(ESBC-DP:CM) mixture raƟos FIG. 1. Total volatile fatty acidity (TVFA) and maximum levels of predominant VFAs for the different mixture ratios tested in the co-digestion study.
the C/N ratio has been considered as the optimum in several literature papers (9,26). However, other authors have proposed lower ranges, indicating that optimum C/N ratio is depending on the waste characteristics (16,27). In the present work, mesophilic anaerobic co-digestion (AcoD) of ESBC-DP and CM was studied, testing the following five mixtures ESBC-DP:CM: 0:100, 25:75, 50:50, 75:25 and 100:0, corresponding to the C/N ratios of 12.5:1, 18.5:1, 23.5:1, 28.5:1 and 33.5:1, respectively. The ESBC-DP used in this work had a very high TS content (874.5 g/kg) and were composed mainly by beet pulp (85%) and molasses (15%). It should be noted that few papers on AD treatment applied to this by-product of the sugar beet industries have been found in literature (6,8). Analysis of the system stability through acidity related parameters Different types of parameters can be used to evaluate the AD process stability. According to literature, pH is one of the most critical variables for biomethanisation process stability. However, the pH value is the result of the acid-base equilibria in the system and, hence, it involves the VFAs productions and the substrates alkalinity. Therefore, in this section, the more important parameters related to the maintaining of pH in the medium have been grouped: pH, total volatile fatty acidity (TVFA), and the two ratios acidity/alkalinity and propionic to acetic acids (HPr/HAc). The pH affects significantly the growth rate of the microorganisms capable to transform the OM into biogas (28). For biomethanisation processes, pH values should be in the range of 6.8e8.5 (10,29). In the present work, during the first days of the process operation, pH dropped below the required range, especially for the reactors with high ESBC-DP content. It can be mainly due to the low alkalinity of ESBC-DP and to the VFAs liberation in the medium (Fig. 1.). As a consequence, pH control with alkali (NaOH, 8M) was necessary once a day during the start-up period, for all
3
reactors, to avoid initial system acidification. However, it was observed that for reactors with mixture ratios of 0:100, 25:75 and 50:50, the pH was stabilized promptly and it remained in the appropriate range, without NaOH addition, after 5, 7 and 8 days, respectively. The short period necessary to reach the suitable pH for the AD process in those reactors with a high CM proportion was mainly related to the high buffer capacity supplied by the livestock substrate (CM) and also the microorganisms variety coming from the ruminant animal tract which are adapted to degrade vegetal fibers (16,17). In Table 2, the initial and final pH values for the different mixture ratios of ESBC-DP and CM are shown. At the end of the process, pH of all reactors was in the required range. Fig. 1 shows the maximum TVFA concentration and levels of the predominant VEAs produced during the assay for the different mixture ratios tested. Acetic acid (HAc) and propionic acid (HPr) were the main VFAs produced along the tests for all mixture ratio reactors. Furthermore, the results point out that the TVFA content increases with increase in the ESBC-DP proportion in the mixtures. Likewise, the maxima for individual VFAs, and especially the maxima propionic acid concentrations, were reached for mixtures with higher ESBC-DP contents. For the reactors 75:25 and 100:0, TVFA started decreasing only after 20 days and 30 days of the assay, respectively. This issue was likely related to the lower buffer capacity of those reactors compared with reactors with high content of CM. As mentioned previously from the characteristics of the substrates, CM has higher nitrogen content and alkalinity. Synergistic effect between carbonaceous wastes and livestock manures was reported to be the reason of neutralizing the VFAs generated, and hence a well operation of the digester (11). The inhibition effect of the propionic acid on AD process is well known (30). Accordingly, acidification was observed in the reactor with the mixture ratio (100:0) producing a delay in biogas production as observed in Fig. 3. However, and despite of the high VFAs accumulation, after a long period of adaptation of the microorganisms, TVFA decreased and biogas was produced. This fact was probably due to the use of an inoculum especially well acclimatized to ESBC-DP treatment as it is coming from a semicontinuous reactor fed with ESBC-DP. On the contrary, the CM addition enhanced the buffer capacity, neutralising the VFAs production and avoiding the acidification and the failure of the system (31,32). Hence, no acidification was produced for reactors with CM proportions above 50%. The CM addition has permitted to maintain an optimal pH for methanogens and to balance the nutrients content in the medium. One of the main criteria to predict the system failure is established by the ratio acidity/alkalinity which permits to analyse the process stability and the balance between liberation of organic acids and the buffer capacity in AD reactors. Fig. 2A shows that for the reactors 0:100 and 25:75 the acidity/alkalinity ratio was in the optimum range, while for reactor 50:50 was slightly higher than 0.4 although limit values for system failure were not exceeded (33). However, when high proportions of ESBC-DP were added to the mixtures, and especially for reactor 100:0, the acidity/alkalinity
TABLE 2. Summarized comparison of the co-digestion process performance for the tested mixture ratios. Mixture ratios (ESBC-DPa:CM)
C/N ratio
SMP (mLCH4/gVSadded)
DSMP /(100:0)
DSMP /(0:100)
(%)
(%)
Methane production rate (LCH4/Lreactor d)
(0:100) (25:75) (50:50) (75:25) (100:0)
12.5 18.5 23.5 28.5 33.5
447.121.8 557.412.2 560.16.4 323.70.9 308.815.6
44.828.7 80.517.1 81.411.5 4.812.7 e
e 24.723.4 25.316.8 27.68.3 30.917.5
0.650.08 1.220.11 0.940.18 0.700.10 0.540.27
a
Removal efficiency (%)
pH
tCOD
VS
Initial pH
Final pH
64.63.5 65.87.2 54.211.8 57.33.8 51.411.2
58.22.4 77.51.6 72.11.8 65.32.1 60.52.4
6.80.1 6.90.1 70.1 70.2 7.10.1
8.30.1 8.20.1 8.30.1 8.20.1 8.20.1
ESBC-DP was composed of 85% of pulp and 15% of molasses.
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ABOUDI ET AL.
(A)
(0:100)
J. BIOSCI. BIOENG.,
(25:75)
(50:50)
(75:25)
(100:0)
acidity/alkalinity raƟo
1.6
1.2
0.8
0.4
0 0
20
40
60
Time (Days)
(B)
(0:100)
(25:75)
(50:50)
(75:25)
(100:0)
10
HPr/HAc raƟo
8
6
4
2
0 0
20
Time (days)
40
60
FIG. 2. Evolution of the ratios acidity/alkalinity (a) and HPr/HAc (b) for the different mixture ratios tested in the co-digestion study.
ratio was over limit values for a long period of the assay before admissible values were reached at the end of the test. Moreover, another widely used parameter to analyse process stability is the ratio HPr/HAc (34e36). In literature, it has been reported that the maximum value for the HPr/HAc ratio is 1.4 and destabilization occurs when this value is exceeded (37). However, other authors have proved that this limit value can vary depending on the characteristics and nature of the substrates and reactor performance (36). Furthermore, Pullammanappallil et al. (34) deduced that conditions that cause inhibition in one system do not necessarily affect to all AD systems. As can be seen in Fig. 2B, in the present work, the HPr/HAc ratio was maintained in the range 0e2 for the reactors 0:100 and 25:75 throughout the test. However, relatively high values were obtained for the reactors 50:50 and 75:25, especially between days 32e42 and 37e48, respectively. Further, for the reactor 100:0, a sharp increase in the HPr/HAc ratio was observed around day 55, reaching a maximum value of 10. Despite of the high values of the HPr/HAc ratio reached in reactors with higher EBSC-DP content (50:50; 75:25 and 100:0), the reactors were able to remove progressively HPr and the ratio started decreasing continuously to achieve more admissible values. Thus, the higher HPr/HAc ratios could be associated to longer periods to reach the VFAs removal and higher lagphase for biogas production. This behaviour can be interpreted by the destabilisation of these reactors due to the uncoupling between metabolic rates of the two involved microorganism groups, the acid producers and the consumers. According to obtained results in the present study, the HPr/HAc ratio of 2 can be considered as an admissible value for a proper
performance of the AD system for ESBC-DP and CM co-digestion at mesophilic conditions. In literature, the specific concentration of HPr leading to AD inhibition is not clear. In fact, a wide variation in HPr concentrations has been considered as inhibitory for the AD process. Thus, Barredo and Evison (38) have considered 1500e2220 mg/L of HPr as the limit range for the AD failure. On the contrary, McCarty and Brosseau (39) have reported that HPr concentrations up to 8000 mg/L can be tolerated by anaerobic digesters if the system is working correctly. The same conclusion was reached by Pullammanappallil et al. (34). In the present study, the maximum HPr concentrations were in the range of 1099e5500 mg/ L and the system was not inhibited since it was able to degrade the accumulated HPr and convert it into biogas as commented previously. Maximum propionic acid concentrations of 1098.8 mg/L, 1935.7 mg/L, 3126.5 mg/L, 35,556.4 mg/L and 5438.36 mg/L were detected for reactors 0:100, 25:75, 50:50, 75:25 and 100:0, respectively. It can be pointed out that propionic acid degradation is a specific activity of acetogenic microorganism populations and its acclimatisation to high HPr concentrations is essential for develop it. For hence, the utilisation in this study of an inoculum well adapted to high HPr concentrations produced by ESBC-DP degradation has been critical. Analysis of the process stability based on the indirect carbon-related parameters As previously commented in the M&M section, and for a better understanding of the relationship between organic acids accumulation, biogas production and the proportions of ESBC dried pellets in the feed of the reactors, a series of parameters have been determined based on those developed by Fdez-Güelfo et al. (25). Fig. 3 shows the evolution of the following parameters: DOC, DAC, ASC and SMP for the different mixture ratios tested. As can be observed in Fig. 3, an initial increase for the new parameter ASC was observed in all reactors, as well as for DOC. These evolutions were due to the hydrolysis of the particulate OM of the wastes, but the ASC increase indicated that transformation in VFAs was not completely coupled initially with hydrolysis rate. The ASC increase was much more pronounced for those mixture ratios with ESBC-DP proportions above 50% (Fig. 3D and E). Additionally, it is clear from Fig. 3 that both the time required to reach the maximum value of ASC as the value of these maxima increased with increasing ESBC-DP content in the mixtures. Thus, time periods of 6, 4, 12, 25 and 26 days were necessary to reach the ASC maxima of 2212.8, 2438.2, 4099.1, 6662.6 and 7367.8 mgC/L for the mixture ratios 0:100, 25:75, 50:50, 75:25 and 100:0 respectively. This fact, revealed that the hydrolysis rate was higher or equal than the acidogenesis rate during the above mentioned periods, leading to the ASC accumulation observed in each case. Similar behavior was reported by Romero-Aguilar et al. (40) when studying AD of the organic fraction of municipal solid wastes for biohydrogen production. Authors reported that when ASC values were higher than DOC, the acidogenic stage was limited or inhibited. However, equilibrium between ASC and the DOC indicates a well coupling of hydrolysis and acidogenesis phases. Subsequently, when the hydrolysis rate decreased, the ASC values dropped quickly. In this case a minor quantity of OM was available to be hydrolysed and solubilized in the medium and the acidogenesis rate was higher than the hydrolysis rate. The ASC decrease was related to its consumption and transformation into VFAs by acidogenic microorganisms. However, the decreasing in ASC was not accompanied with an increase in DAC but rather with the increase in methane production. This fact is indicative that acidogenic and methanogenic activities were coupled and well balanced and VFAs were transformed into methane as soon as they were formed. Only a slight deviation
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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FIG. 3. Specific methane production (expressed as mLCH4/gVSadded), dissolved organic carbon (DOC), dissolved acid carbon (DAC) and acidogenic substrate as carbon (ASC) for the different mixture ratios tested in the co-digestion study.
between times required to reach the maxima of ASC and DAC was observed for the reactor 100:0. Moreover, in this case, biogas production started once DAC decreased. In short, the evolution of the reactors is highly related with the ESBC-DP proportion in the mixture. Thus, the reactors with minor contents in ESBC-DP (the mixtures 0:100 and 25:75) showed a similar behaviour in the ASC trend, and the different stages of the process e hydrolysis, acidogenesis and methanogenesis e were well coupled. On the contrary, the reactors with higher contents in ESBC-DP (the mixtures 75:25 and 100:0) have also shown a similar evolution. In these cases, decoupling was observed between hydrolytic and acidogenic stages, leading to ASC accumulations.
However, inhibition was not observed for the methanogenic stage. The reactor 50:50 shows an intermediate comportment. Enhancement in the AcoD efficiency: OM removal and methane production The main parameters to analyse the performance of the mesophilic batch anaerobic digesters used in this study are shown in Table 2. According to the obtained results, the higher tCOD removal was obtained for the reactors 0:100 and 25:75 reaching values above 64%. For the rest of reactors, tCOD removal ranged of 51e58%. Regarding the effect of the co-digestion on methane production, the obtained results showed that all the tested mixture ratios were
Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005
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better than the mono-digestion of ESBC-DP. Table 2 shows that increments of specific methane production (SMP, expressed as mLCH4/ gVSadded) with respect to the mixture ratio 100:0 were of 80.5% and 81.4% for the mixtures 25:75 and 50:50 respectively, while only a slight increase (4.8%) was obtained for the mixture 75:25. Methane production rates (MPR, expressed as LCH4/Lreactor d) are shown in Table 2. As can be observed, the highest MPRs were observed for reactors 25:75 and 50:50 of about 1.22 0.11 LCH4/ Lreactor d and 0.94 0.18 LCH4/Lreactor d, respectively. Individual digestion of CM and ESBC-DP showed lower MPRs of 0.65 0.08 LCH4/Lreactor d and 0.54 0.27 LCH4/Lreactor d, respectively. Furthermore, analysing the increments of SMP with respect to the mixture 0:100, which corresponds to the digestion of CM as a sole substrate, results showed that the mixtures 25:75 and 50:50 improved the SMP in a 24.7% and 25.3%, respectively. In short, the addition of ESBC-DP to CM above 50% affected negatively the methane production with respect to anaerobic mono-digestion of CM. Another important issue that supports the feasibility and the improvement due to the ESBC-DP co-digestion with CM is related to the required time for process start-up. From the results of the SMP in Fig. 3, it can be concluded that the lag-phase period for methane production can be reduced significantly when the ESBC-DP was codigested with CM. Besides, the lag-phase period was lower for the greater proportion of CM in the mixture. The minimum lag-phase period of 6 days was observed for the mixture 25:75, instead of 37 days for the digestion of ESBC-DP individually or 9 days for the CM mono-digestion. The lag-phase is the time required by microorganisms for their adaptation to the environment conditions. Thus, considering the complex nature of ESBC-DP (lignocellulosic-type substrate), it can be expected that the microorganisms needed a longer adaptationtime for mixtures with higher ESBC-DP proportions. In this work it has been observed that lag-phase was shortened by CM addition, likely due to that livestock wastes provided microorganisms and all the macro and micro nutrients required for the microbial growth from the rumen (16,17), creating a synergistic effect in the medium (11). Moreover, the big lag-phase periods observed for the reactors with ESBC-DP proportions above 50% were also related to the higher levels of the ASC accumulated in the medium in these tests. Hence, the delay in methane production was associated to the transformation of ASC into organic acids (delay in acidogenesis stage) because no VFAs accumulation was observed. Once the systems were adapted to the high ASC concentrations, the organic acid production started at the same time as its transformation into methane as discussed in the previous section. This behaviour indicates a well coupling of the acidogenic and methanogenic phases. A comparison with other works focusing on co-digestion of similar substrates was reported in a previous paper (6), and recently published works were also compared to the present research. As a conclusion, it can be pointed out that the results obtained in the present study about individual digestion of ESBCDP were in the inferior limit of those obtained by other authors (7), and quite comparable to several studies (6,19). However, the optimal result obtained for co-digestion process in the current research were in the superior limit of other works. Fang et al. (7), obtained SMPs of 490, 500 and 240 mlCH4/gVSadded, respectively for AD of sugar beet leaves, tops and sugar beet pulp. Similarly, Gissén et al. (41) studied AD of sugar beet by-product and obtained a SMP of 419 mlCH4/gVSadded. The investigation performed by Montañes et al. (22) using sugar beet pulp lixiviation and sewage sludge showed a SMP of 544 mlCH4/gVSadded. Aboudi et al. (6) obtained a SMP of 494 mlCH4/gVSadded from AcoD of ESBC with PM. In another attempt, Aboulenein et al. (42) obtained very high SMP of 695 mlCH4/gVSadded when studying AcoD of CM with agricultural wastes (coconut, cassava pulp, coffee grounds).
J. BIOSCI. BIOENG., According to the discussed parameters trends, the mixtures 25:75 and 50:50 had been the best mixtures tested in this study with high methane production and process stability while ESBC-DP addition above 50% affected negatively the AcoD process. ACKNOWLEDGMENTS This work was supported by the Spanish Ministry of Economy and Finance-ERDF for the IþDþI project (CTM2013-43938-R) and co-funded by the MICINN-Spain (UNCA08-1E-035 Project). The authors acknowledge to the University of Cadiz (Spain) for the Scholarship UCA-2010-063PU/EPIF-FPI-A/BC and the Agrifood Campus of International Excellence (Ceia3). The authors also wish to thank the Spanish Ministry of Science and Innovation (PROBIOGAS Project PS-120000-2007-6). References 1. Sawatdeenarunat, C., Surendra, K. C., Takara, D., Oechsner, H., and Khanal, S. K.: Anaerobic digestion of lignocellulosic biomass: challenges and opportunities, Bioresour. Technol., 178, 178e186 (2015). 2. Weiland, P.: Biogas production: current state and perspectives, Appl. Microbiol. Biotechnol., 85, 849e860 (2010). 3. Ward, A. J., Hobbs, P. J., Holliman, P. J., and Jones, D. L.: Optimisation of the anaerobic digestion of agricultural resources, Bioresour. Technol., 99, 7928e7940 (2008). 4. Anwar, Z., Gulfraz, M., and Irshad, M.: Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review, J. Radiat. Res. Appl. Sci., 7, 163e173 (2014). 5. Nishio, N. and Nakashimada, Y.: Recent development of anaerobic digestion processes for energy recovery from wastes, J. Biosci. Bioeng., 103, 105e112 (2007). 6. Aboudi, K., Álvarez-Gallego, C. J., and Romero-García, L. I.: Improvement of exhausted sugar beet cossettes anaerobic digestion process by co-digestion with pig manure, Energy Fuels, 29, 754e762 (2015). 7. Fang, C., Boe, K., and Angelidaki, I.: Anaerobic co-digestion of by-products from sugar production with cow manure, Water Res., 45, 3473e3480 (2011). 8. Hutnan, M., Drtil, M., and Mrafkova, L.: Anaerobic biodegradation of sugar beet pulp, Biodegradation, 11, 203e211 (2000). 9. Wang, X., Yang, G., Feng, Y., Ren, G., and Han, X.: Optimizing feeding composition and carbon-nitrogen ratios for improved methane yield during anaerobic co-digestion of dairy, chicken manure and wheat straw, Bioresour. Technol., 120, 78e83 (2012). 10. Zoetemeyer, R. J., van den Heuvel, J. C., and Cohen, A.: pH influence on acidogenic dissimilation of glucose in an anaerobic digestor, Water Res., 16, 303e311 (1982). 11. Pagés-Díaz, J., Pereda-Reyes, I., Taherzadeh, M. J., Sárvári-Horváth, I., and Lundin, M.: Anaerobic co-digestion of solid slaughterhouse wastes with agroresidues: synergistic and antagonistic interactions determined in batch digestion assays, Chem. Eng. J., 245, 89e98 (2014). 12. Mata-Alvarez, J., Macé, S., and Llabrés, P.: Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives, Bioresour. Technol., 74, 3e16 (2000). 13. Cuetos, M. J., Fernández, C., Gómez, X., and Morán, A.: Anaerobic co-digestion of swine manure with energy crop residues, Biotechnol. Bioprocess Eng., 16, 1044e1052 (2011). 14. Hartmann, H. and Ahring, B. K.: Anaerobic digestion of the organic fraction of municipal solid waste: influence of co-digestion with manure, Water Res., 39, 1543e1552 (2005). 15. Tian, H., Duan, N., Lin, C., Li, X., and Zhong, M.: Anaerobic co-digestion of kitchen waste and pig manure with different mixing ratios, J. Biosci. Bioeng., 120, 51e57 (2015). 16. Kivaisi, A. K. and Eliapenda, S.: Application of rumen microorganisms for enhanced anaerobic degradation of bagasse and maize bran, Biomass Bioenergy, 8, 45e50 (1995). 17. Hindrichsen, I. K., Wettstein, H. R., Machmüller, A., Knudsen, K. E. B., Madsen, J., and Kreuzer, M.: Digestive and metabolic utilisation of dairy cows supplemented with concentrates characterised by different carbohydrates, Anim. Feed Sci. Technol., 126, 43e61 (2006). 18. Ohuchi, Y., Ying, C., Lateef, S. A., Ihara, I., Iwasaki, M., Inoue, R., and Umetsu, K.: Anaerobic co-digestion of sugar beet tops silage and dairy cow manure under thermophilic condition, J. Mater. Cycles Waste Manage., 17, 540e546 (2014). 19. Alkaya, E. and Demirer, G. N.: Anaerobic mesophilic co-digestion of sugar-beet processing wastewater and beet-pulp in batch reactors, Renew. Energy, 36, 971e975 (2011).
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Please cite this article in press as: Aboudi, K., et al., Evaluation of methane generation and process stability from anaerobic co-digestion of sugar beet by-product and cow manure, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.10.005