- Email: [email protected]
Enhanced methane production from pig manure anaerobic digestion using fish and biodiesel wastes as co-substrates
Bioresource Technology 123 (2012) 507–513
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced methane production from pig manure anaerobic digestion using fish and biodiesel wastes as co-substrates Leticia Regueiro a, Marta Carballa a,⇑, Juan A. Álvarez b, Juan M. Lema a a b
Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Rúa Lope Gómez de Marzoa s/n, 15782 Santiago de Compostela, Spain AIMEN Technological Center, C/Relva, 27 A – Torneiros, 36410 Porriño Pontevedra, Spain
h i g h l i g h t s " Fish and biodiesel waste were used as co-substrates in pig manure anaerobic digestion. " Both co-substrates improved methane yield but caused VFA and ammonium accumulation. " Shorter HRT and FW < 10% in the feeding allow to control ammonium inhibition. " Biodiesel waste co-digestion requires feeding shares < 6% and/or fed-batch operation. " The poorer the co-digester operation, the higher the Methanosarcina/Methanosaeta ratio.
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 26 July 2012 Accepted 28 July 2012 Available online 7 August 2012 Keywords: Agroindustrial wastes Ammonia Co-digestion Methanosarcina Volatile fatty acids
a b s t r a c t Co-digestion of pig manure (PM1) with fish (FW2) and biodiesel waste (BW3) was evaluated and compared with sole PM digestion. Results indicated that co-digestion of PM with FW and/or BW is possible as long as ammonium and volatile fatty acids remained under inhibitory levels by adjusting the operating conditions, such as feed composition, organic loading rate (OLR) and hydraulic retention time (HRT). PM and FW codigestion (90:10 and 95:5, w/w4) was possible at OLR of 1–1.5 g COD/L d, resulting in biogas production rates of 0.4–0.6 L/L d and COD removal efficiencies of 65–70%. Regarding BW, good results (biogas production of 0.9 L/L d and COD elimination of 85%) were achieved with less than 5% feeding rate. Overall, operating at the same OLR, the biogas production and methane content in the co-digester was higher than in the only PM digester. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In the last years, anaerobic digestion of animal wastes has been promoted in order to avoid the uncontrolled emissions of CH4 during storage (Novak and Fiorelli, 2010). Pig manure (PM5) can be an excellent base substrate for anaerobic digestion due to its inherent buffering capacity and high content of a wide range of nutrients required for the development of anaerobic microorganisms. However, PM has a low biogas yield, around 20–30 m3/ton (Angelidaki and Ellegaard, 2003), and high ammonium concentrations (2–3 g N– ⇑ Corresponding author. Tel.: +34 881 816020; fax: +34 881 816702. E-mail addresses: [email protected] (L. Regueiro), marta. [email protected] (M. Carballa), [email protected] (J.A. Álvarez), [email protected] (J.M. Lema). 1 PM, pig manure; 2 FW, fish waste; 3 BW, biodiesel waste; 4 w/w, wet weight basis; 5 PM, pig manure; 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.109
NH4+/L). Consequently, PM is preferably co-digested with high carbon content wastes, on one hand, to improve the C/N ratio (Hartman and Ahring, 2006), and on the other hand, to increase the biogas production, essential for the plant’s economy. It has been shown that bioenergy production in farm biogas plants could be enhanced by 80–400% by using organic wastes and by-products as co-substrates (Braun and Wellinger, 2003; Weiland, 2010). Despite the wellknown reported co-digestion benefits, such as optimum humidity and C/N ratio or inhibitory substances dilution (Mata-Álvarez et al., 2000), it is not clear whether some substrates have adverse impact when they are co-digested with another waste (Callaghan et al., 2002). Therefore, it is critical to obtain an optimal mixture of the available co-substrates as well as the optimum operating conditions which allow high biogas yields without compromising the stability of the process (Alvarez et al., 2010). Fish and shellfish canning industry is an important sector in Galicia (NW of Spain), with around 65% of the total Spanish production and representing 45% of the Galician factories and 67% of the jobs (Garcia et al., 2003). This sector generates different
508
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
solid wastes with a wide range of characteristics depending on the raw material processed (tuna, mussel, sardine, mackerel, etc.). In general, fish wastes (FW6) are protein-rich substrates, although they also contain important lipids. Protein-rich materials have a fast biomethanation, but their degradation products (ammonium) can inhibit the process as well (Chen et al., 2008). Ammonium inhibition is directly related to the concentration of the undissociated form (NH3) or free ammonia (FA7), thus becoming more important at high pH levels. The inhibitory FA concentration varies depending on operational parameters such as origin of inoculum, substrate, pH and temperature (Alvarez and Liden, 2008). The reported FA inhibitory concentrations for mesophilic conditions range from 25 to 140 mg N-FA/L, whereas during the thermophilic digestion of cattle manure, higher values (around 390–700 mg N-FA/L) were tolerated after an initial acclimation period (Guerrero et al., 1997). Biodiesel fuels have recently drawn much attention given that they have various advantages over petroleum-based fuels (Ito et al., 2005). The biodiesel production is carried out by catalyzed transesterification with alcohol (usually methanol). Beside desired methylesters, this process also generates few other products, including crude glycerol or biodiesel waste (BW8), oil-pressed cakes and washing water. BW is easily separated from the aqueous phase and it is composed mainly of glycerol. In many occasions, it also contains a significant fraction of lipids due to inefficient separation systems. It is estimated that 1 kg of crude glycerol is generated per 9 kg of biodiesel produced (Dasari et al., 2005). The important increase in the biodiesel production of the last years has resulted in crude glycerol surplus that implied a dramatic 10-fold decrease in biodiesel waste prices (Yazdani and Gonzalez, 2007), and consequently, crude glycerol is often regarded as a waste stream with an associated disposal cost. This biodiesel residue is readily digestible and can be easily stored over a long period, making it an ideal co-substrate for anaerobic digestion (Ma et al., 2008), whereas its digestion as sole substrate is not viable as no nitrogen would be available for microbial populations (Robra et al., 2010). Lipid-rich materials are known to have high methane production potentials (Hansen et al., 1999), but their degradation products, the long-chain fatty acids (LCFA), are known to be inhibitors of methanogenic microorganisms (Pereira et al., 2004). Besides, operational instabilities related to sludge flotation and washout are also reported (Jeganathan et al., 2006). The aim of this work was to evaluate the use of fish waste and biodiesel waste as co-substrates to enhance the mesophilic anaerobic digestion of pig manure at laboratory scale. The effect of feeding mixture on process performance and microbial community composition was investigated. The results obtained in the co-digestion systems were compared with those obtained in a reactor treating only pig manure.
2. Methods 2.1. Wastes and inoculum PM was taken from a sewer of a 150-pig fattener and sow farm, which collects both feces and urine. PM samples were homogenized, sieved to 2 mm and stored at 4 °C until use to minimize decomposition. Different batches of PM were used throughout the experiment (>200 days) due to the impossibility of storing the total amount required. FW was delivered by a canning industry and it consisted of heads, tails, bones and viscera of tuna fish. FW was homogenized 6 7 8
FW, fish waste; FA, free ammonia; BW, biodiesel waste;
by grinding and stored at 20 °C. BW was taken from a biodiesel factory and was stored at 4 °C without pre-treatment. It contained mainly glycerol produced in the transesterification. One batch of FW and BW was sufficient for the complete study. Anaerobic granular biomass from an internal circulation reactor treating brewery wastewater was used as inoculum, with an initial in-reactor biomass concentration of about 10 g VSS/L. 2.2. Anaerobic reactors Experiments were carried out in three continuous stirred tank reactors (160 rpm, Heidolph RZR 2041), two co-digesters and one only-PM digester, constructed in methacrylate and with a working volume of 9.2 L, approximately. Reactors were operated at 35 °C by hot water recirculation. The applied feedstock mixtures were prepared every 2–3 days, diluted with tap water according to the applied organic loading rate (OLR), and stored at 4 °C prior to use. The digesters were fed manually after an equivalent volume of digester mixed liquor was removed. Temperature, pH, stirring speed and biogas production were monitored on-line. Other physico-chemical parameters (solids, chemical oxygen demand (COD), alkalinity, volatile fatty acids (VFA) and ammonium) were measured twice per week. 2.3. Operational strategy The operation of the two co-digesters was identical. They were started-up with a mixture of PM-FW-BW (84:5:11 in wet weight (w/w) basis), which was the optimum mixture obtained from linear programming (Alvarez et al., 2010), at an OLR of 0.5 g COD/L d and a hydraulic retention time (HRT) of 40 d. After the start-up (day 0–19), the operation of the co-digesters can be divided in three periods according to the feeding blend, HRT and OLR applied. In period I (days 20–59), the reactors were fed with a mixture of PM and FW (90:10, w/w) at OLR of 1 g COD/L d and HRT of 35 d. In period II (days 60–115), the percentage of FW was decreased to 5% and the OLR and HRT were increased to 1.5 g COD/L d and decreased to 30 d, respectively. In addition, three pulses of BW (5 g COD/L) were added to the reactors on days 80, 90 and 100. In the last period (period III, days 116–200), FW was replaced by BW in order to prevent ammonia inhibition, the OLR was increased to 2 g COD/L d and the HRT was reduced to 25 d. In all periods, the feeding mixture was diluted with tap water to attain the proper OLR. The start up of the only-PM digester with OLR of around 0.5 COD/L d and HRT of 20 d took 36 days. In period I (days 37–89), the OLR was increased up to 1 g COD/L d and the HRT was slightly lowered to 18 d in order to reach the target OLR, since the COD of PM was lower in this period. The OLR was further increased to 1.5 and 2 g COD/L d and the HRT was decreased to 15 and 12 d in periods II (days 90–104) and III (days 105–150), respectively. Since different batches of PM were used along the experiment (with different COD concentrations), dilution with tap water was not always required to attain the established OLR. 2.4. Analytical methods pH, COD, total solids (TS), volatile solids (VS), total suspended solids (TSS), volatile suspended solids (VSS), total Kjeldahl nitrogen (N-TKN), ammonium (N–NH4+), TA (total alkalinity) and PA (partial alkalinity) were performed following standard methods (APHA, 1995). Biogas production was measured online by Ritter milligascounters (Dr. Ing. Ritter Apparatebau GmbH, Bochum, Germany) and biogas composition was analyzed by gas chromatography (HP, 5890 Series II). VFA (acetic, propionic, i-butyric, n-butyric, i-valeric and n-valeric) were analyzed by gas
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
chromatography (HP, 5890A) equipped with a Flame Ionization Detector (HP, 7673A). Total lipids content was determined using the standard Soxhlet method (APHA, 1995) and protein concentration was calculated from the organic nitrogen content. Carbohydrates were estimated as the remaining fraction of VS after proteins and lipids were subtracted. FA concentration was calculated using the NH4+–NH3 equilibrium constant (Ka), N–NH4+ concentration (g/L), pH and temperature (T, °C), according to equations 1 and 2 (Cuetos et al., 2008):
NH3 ¼
NHþ4
ð1Þ
1 þ 10ðpKapHÞ
pKa ¼ 4 108 T3 þ 9 105 T2 0:0356T þ 10:072
ð2Þ
2.5. Fluorescent in situ hybridization (FISH) Active bacterial and archaeal populations were identified and semi-quantified by the FISH technique. Fresh biomass samples were collected from the reactors on days 70, 90, 110 and 200, disrupted and fixed with 4% paraformaldehyde solution according to the procedure described by Amann et al. (1995). Hybridization was performed at 46 °C for 90 min adjusting the formamide concentrations for each probe. The probes used were: Eub338mix (Bacteria), Arc915 (Archaea), Ms821 (Methanosarcina), Mx825 (Methanosaeta). All the details of each probe (formamide percentage, sequence and target organism) can be found in the probeBase database (http://www.microbial-ecology.net/probebase/). All probes were 50 labeled with the fluorochromes FITC and Cy3. Fluorescence signals were recorded with an acquisition system (Coolsnap, Roper Sicientific Photometrics) coupled to an Axioskop 2 epifluorescence microscope (Zeiss, Germany). DAIME program (Daims et al., 2006) was used to make the semi-quantitative counting with at least six photos taken per 20 lL of fixed sample (108–109 cells per mL). 3. Results and discussion 3.1. Wastes characterization Table 1 shows the physico-chemical characterization of the substrates (PM, FW and BW) used in this study. The three substrates had neutral pH values (between 6.9 and 7.3). PM had low TS (17 g/kg), VS (12 g/kg) and COD (30 g/kg) levels compared to FW (370 g TS/kg, 270 g VS/kg and 410 g COD/kg) and BW
Table 1 Physico-chemical characteristics (average values) of pig manure, fish waste and biodiesel waste. Parameter
Pig manurea
Fish waste
Biodiesel waste
pH Density (kg/L) TS (g/kg) VS (g/kg) CODtotal (g O2/kg) CODsoluble (g O2/kg) N-TKN (g N/kg) N–NH4+ (g N/kg) Total alkalinity (g CaCO3/kg) Proteins (g/kg) Lipids (g/kg) Carbohydrates (g/kg) COD/N ratio
6.9 ± 0.2 1.0 ± 0.0 17.3 ± 4.5 11.7 ± 5.3 29 ± 12 15.3 ± 7.1 3.3 ± 0.6 3.1 ± 0.4 7.7 ± 1.3 1.1 ± 0.2 1.5 ± 0.3 9.2 ± 3.8 8.9 ± 1.3
7.1 1.1 369 270 410 – 34 0.7 0.4 206 28 36 12.2
7.3 1.0 950 938 1390 – 0.2 0 0 1.2 77.3 922 7315
TS, total solids; VS, volatile solids; COD, chemical oxygen demand; N-TKN, total Kjeldahl nitrogen; N–NH4+, ammonium. a Standard deviations are only shown for pig manure since several batches of this substrate were used along the experiment.
509
(950 g TS/kg, 940 g VS/kg and 1390 g COD/kg), but high alkalinity (7.7 g/kg). As expected, the highest nitrogen concentration was found in FW (34 g N-TKN/kg), while it was negligible in BW. In contrast, BW had a high content of lipids (77.3 g/kg) and of easily biodegradable carbohydrates (almost 925 g/kg). In summary, PM provides moisture and alkalinity, FW provides mainly nitrogen from proteins and also lipids but in a minor proportion (mainly from fishbone fiber and cartilage) and BW provides easily degradable COD (carbohydrates) and lipids. 3.2. Continuous pig manure anaerobic co-digestion Two co-digesters were operated for 200 days at identical conditions (duplicates). The performance of the two co-digesters was very comparable, and therefore, only data from one codigester are shown in figures and tables. pH and biomass concentrations remained at 7.2–8.2 and 5–7 g VSS/L, respectively, during the whole experiment (data not shown). Fig. 1 shows the performance of one co-digester in terms of OLR and biogas production (Fig. 1A), COD concentrations in influent and effluent (Fig. 1B), VFA levels (Fig. 1C) and ammonium and FA concentrations (Fig. 1D). At the end of the start-up period (day 19), the biogas production reached 0.22 L/L d (Fig. 1A), with methane content of 50%, and the COD removal efficiency was around 61%. During this period, acetic acid was consumed, but propionic acid remained in values around 800 mg/L (Fig. 1C). This was probably due to the relatively high volumetric percentage of BW in the feeding mixture (11%), since BW is considered as rapidly biodegradable organic substrate, which generates large amounts of VFA (Astals et al., 2011). Several authors (Amon et al., 2006; Astals et al., 2011) have shown that 4–6% (w/w) of BW in the feeding is considered to be the limiting concentration to maintain a stable anaerobic digestion process. Moreover, the propionic to acetic acid ratio at the end of the start-up was about 3.8 and it has been reported that a propionic to acetic acid ratio greater than 1.4 is a hint of immediate digester failure (Pullammanappallil et al., 2001). Thus, in period I, BW was eliminated from the feeding mixture. In the first days of period I (days 20–30), when the co-digester was fed with a mixture of PM and FW (90:10, w/w), the VFA content decreased to less than 0.2 g/L, despite the OLR doubling i.e. from 0.5 to 1 g COD/L d. This fact suggests that the elimination of BW from the feeding mixture had a satisfactory effect on process stability. Consequently, the biogas production and the COD removal increased to 0.43 L/L d and 70%, respectively. But from day 30 on, ammonium concentrations started rising (Fig. 1D) as a result of the digestion of the proteins present in FW and the high ammonium levels in PM (3 g N–NH4+/kg, Table 1). Although no accumulation of VFA was observed (Fig. 1C), total COD concentrations in the effluent increased from 9 to 11.5 g/L (Fig. 1B) and biogas production decreased to 0.25 L/L d (Fig. 1A) by the end of this period. To attenuate the ammonium accumulation, the percentage of FW in the feeding mixture and the HRT were decreased in period II in order to promote ammonium wash out. As expected, ammonium levels decreased during the first days of period II, but from day 80 on, they increased again (Fig. 1D). The change in the PM stock (with higher ammonium concentrations) combined with the higher OLR applied in this period (1.5 g COD/L d, Table 2) provoked this increase in the ammonium levels in the reactors. In period II, three pulses of BW (5 g COD/L) were performed on days 80, 90 and 100, following the strategy proposed by Cavaleiro et al. (2009) to achieve efficient methane yields from lipid-rich substrates. This strategy consists of the combination of lipid-rich substrate feeding periods (LCFA accumulation) with non-feeding periods (degradation of accumulated LCFA). These authors
510
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Fig. 1. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (BW pulses in period II were not considered in influent COD (CODin)) (B), volatile fatty acids (VFA) concentration (C) and ammonium and free ammonia (FA) concentrations (D) in one co-digester. Arrows in Fig. 1A indicate BW pulses of 5 g COD/L on days 80, 90 and 100.
Table 2 Operational parameters of the lab-scale anaerobic co-digester and only-pig manure digester during the different operational periods (values shown corresponded to steady-state conditions of each period).
a
Type reactor
Period
PM-FW-BW (% w/w)a
Duration (days)
OLR (g COD/L d)
HRT (d)
pH
CH4 (%)
COD removal (%)
Biogas (L/L d)
Co-digester
Start-up Period I Period II Period III
84–5-11 90–10-0 95–5-0 95–0-5
0–19 20–59 60–115 116–200
0.5 1.0 1.5 2.0
40 35 30 25
7.4 ± 0.2 7.6 ± 0.3 8.0 ± 0.2 7.7 ± 0.2
50 ± 2 57 ± 3 59 ± 2 60 ± 3
60.8 ± 0.3 69.6 ± 1.2 65.7 ± 0.8 78.5 ± 0.7
0.22 ± 0.01 0.43 ± 0.04 0.59 ± 0.05 0.91 ± 0.06
Digester
Start-up Period I Period II Period III
0–36 37–89 90–104 105–150
0.6 1.0 1.5 2.0
20 18 15 10
7.6 ± 0.2 7.9 ± 0.2 8.0 ± 0.2 8.1 ± 0.1
56 ± 4 52 ± 7 52 ± 5 55 ± 6
40.2 ± 0.4 37.5 ± 0.9 40.1 ± 2.0 52.6 ± 3.7
0.15 ± 0.02 0.25 ± 0.03 0.40 ± 0.01 0.67 ± 0.10
PM
w/w: wet weight.
reported that at least two cycles of accumulation-degradation of LCFA were necessary before continuous operation The positive effect of BW pulses on biogas production was clear (Fig. 1A), but
the % of pulse COD converted into methane in relation to the maximum theoretical methane production (taking into account 0.35 m3 CH4/kg CODconverted) decreased with each consecutive
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
pulse (52.1%, 26.8% and 24.2%), probably as a result of the increase in VFA levels after the first pulse addition (Fig. 1C). The major components of the VFAs were acetic and propionic acid, which reached values up to 2.5 g/L and 2.0 g/L, respectively. The increased VFA levels also resulted in higher effluent COD concentrations (Fig. 1B), and consequently, the COD removal efficiency decreased from 70% (before pulses) to 66% (after pulses). By the end of period II, VFA and the COD levels lowered, but did not reach the original values (Fig. 1B and C), indicating that organic compounds related to BW, such as alcohols or LCFA, were accumulated into the co-digesters. In contrast, the biogas production was not inhibited, suggesting that the degradation of PM:FW mixture was not affected and just the COD from pulses was accumulated. By the end of period II, the ammonium levels were still high (2 g N–NH4+/L), which combined with high pH values (around 7.8, data not shown) resulted in FA levels around 260 mg N–NH3/L, which might be inhibitory for anaerobic communities (Chen et al., 2008). Furthermore, FA levels were much more dependent on pH variations than on the ammonium concentration, because between days 106 and 112, FA increased from 125 to 260 mg N–NH3/L, when the pH varied from 7.8 to 8.1, despite the decrease of the ammonium levels from 2.2 to 2 g N–NH4+/L (Fig. 1D). Consequently, FW was replaced by BW in period III. In this case, the percentage of BW in the mixture (5%) did not exceed the 6% that Amon et al. (2006) set as maximum for a good performance. Besides, HRT was decreased to 25 d in order to promote ammonium washout. These changes led to a drop in ammonium and FA concentrations, which stabilized at 1300 mg N–NH4+/L and 100 mg N–NH3/L, respectively, by the end of the experiment (Fig. 1D). In period III, biogas production increased as a consequence of the higher OLR applied (Fig. 1A). However, from day 130, it went down due to the accumulation of propionic acid (Fig. 1C), resulting on 0.3 L/L d on day 150. To surpass this inhibition, the co-digesters were not fed between days 151 and 160 and propionic acid was consumed giving rise to a recovery in biogas production rates. After this event, the co-digesters operated stably for 40 days, with biogas production rates of 0.9 L/L d and COD removal efficiencies of almost 80%, which emphasizes the high biodegradability of BW. The microbiology of the co-digesters in the last two operational periods was studied with the FISH technique (Supplementary material (SM), Fig. SM 1). A rough estimation of the relative shares of each main population indicated that half of the active population was Archaea and the other half was Bacteria. This ratio did not vary, even when the feeding mixture changed as happened between days 100 and 120. The relative abundance of archaeal populations (Methanosaeta and Methanosarcina) present was correlated with the co-digester performance, particularly with VFA concentrations. Before the pulses (day 70), the predominant archaeal population was Methanosaeta (Fig. SM-1A), demonstrated by the presence of the characteristic shape of this population in tubular sheath. Methanosaeta are well known as the most abundant acetoclastic methanogens in bioreactors with low VFA and ammonium levels (Karakashev et al., 2008), as occurred before the pulses. After the three pulses (day 90), this population disappeared almost completely and the niche was filled by Methanosarcina, which displayed the individual coccoid cells shape forming aggregates similar to a bunch of grapes (Fig. SM-1B). Methanosarcina is able to use both the acetoclastic and the hydrogenotrophic pathways, thus being more tolerant to methanogenesis inhibitors compared to Methanosaeta (Liu et al., 2011). Its presence in anaerobic reactors is often associated with the deterioration of reactor performance (Blume et al., 2010; Hori et al., 2006). Consequently, the appearance of this archaeal population is probably related to the VFA accumulation that occurred between days 85 and 110 (Fig. 1C). On day 120 (Fig. SM-1C), when
511
the co-digester was recovering from inhibition (VFA concentration was decreasing), the two methanogenic populations coexisted in the reactor. At the end of the experiment (day 200), when the co-digester showed a good stable performance, the methanogenic population was again mainly Methanosaeta (Fig. SM-1D). These variations in Methanosaeta and Methanosarcina populations were semi-quantified by using the probes Mx825 and Ms821, respectively. Methanosaeta fractions (with respect to total archaeal population) were 95%, 0%, 45% and almost 100% on days 70, 90, 110 and 200, respectively, while those of Methanosarcina were 0%, 100%, 40% and 0%, respectively. 3.3. Continuous pig manure anaerobic digestion pH and biomass concentrations remained at 7.4–8.3 and 5–8 g VSS/L, respectively, during the whole experiment (data not shown). Fig. 2 shows the results of the operation of the reactor treating PM as a sole substrate. During the start-up (day 0–36), biogas production stayed at around 0.15 L/L d (Fig. 2A) with methane content of 56%, leading to average COD removal efficiencies of 40% (Fig. 2B). VFA concentrations were negligible (Fig. 2C) and ammonium levels remained below 1.2 g N–NH4+/L (Fig. 2D). The increase in the OLR from 0.6 to 1 g COD/Ld in period I derived in greater biogas production rates (0.25 L/L d, methane content of 52%), but the COD removal efficiencies remained at lower values (around 38%). The accumulation of ammonium observed at the end of the start-up continued and stabilized at around 3 g N–NH4+/L (Fig. 2D). The latter combined with the accumulation of VFA between days 65 and 80 (Fig. 2C) provoked a drop on the biogas production, and consequently, on the COD removal efficiencies (Fig. 2B). Although the reactor recovered quite quickly from VFA accumulation, ammonium concentrations were still high (Fig. 2D), and therefore, the HRT was decreased to 15 days in period II. This modification combined with a higher COD content in PM yielded an increase in the OLR to 1.5 g COD/L d. Consequently, biogas production and COD removal efficiencies increased to 0.40 L/L d and 40%, respectively. Since ammonium levels did not decrease, the HRT was further lowered to 10 days on day 115 (period III). Despite the fluctuations in the OLR (1.6–2.2 g COD/L d, Fig. 2A) caused by variations in COD content of PM, biogas production remained quite constant at 0.7 L/L d (Fig. 2A). By the end of the experiment, VFA concentrations were negligible (Fig. 2C), but ammonium and FA stayed at around 2.5 g N–NH4+/L and 250 mg N–NH3/L, respectively (Fig. 2D). 3.4. Improved pig manure anaerobic digestion by using FW and BW Table 2 shows a comparison of the results obtained in the co-digester and in the only-PM digester at steady-state conditions during each operational period. Operating at the same OLR, the biogas production and methane content in the co-digester were higher than in the only-PM digester. More specifically, biogas production was improved by 47%, 72%, 48% and 36% at OLR of 0.5, 1, 1.5 and 2 g COD/L d, respectively. Moreover, the COD converted into methane was much higher in the co-digestion process, reaching values near to 79% (period III), while in the digestion process the maximum value was 53% (period III). An additional advantage of the co-digestion process was the ammonium and FA control in the reactors. The highest values achieved in the co-digester (2 g N–NH4+/L and 250 mg N–NH3/L) were around 1.5-fold lower than those in the only-PM digester (3.2 g N–NH4+/L and 320 mg N–NH3/L). Although anaerobic microorganisms can adapt to relatively high FA levels, some studies suggested that microorganisms adapted to the anaerobic digestion of PM were inhibited at FA concentrations around 0.7–1.1 g N–NH3/L (Nielsen and Angelidaki, 2008).
512
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Fig. 2. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (B), volatile fatty acids (VFA) concentration (C) and ammonium and free ammonia (FA) concentrations (D) in the pig manure anaerobic reactor.
The main problems associated to FW and BW were ammonium and VFA accumulation, respectively. The first can be surpassed by working at low OLR as in period II (around 1.5 g COD/L d) and/or at shorter HRT to promote ammonium washout, and the second one can be solved by adapting the feeding strategy of BW. In this study, two alternatives were evaluated: pulses in period II and continuous operation in period III. Not good results were obtained with the pulses, probably due to their frequency and COD concentration, while the continuous operation with BW percentages below 6% in the feeding mixture derived a stable performance with good biogas production (0.91 L/L d), which is consistent with results obtained by other authors (Astals et al., 2012; Robra et al., 2010). 4. Conclusions Anaerobic co-digestion of PM with FW or BW evidenced an upgrade with respect to sole PM digestion, not only in terms of increased biogas production, but also on process stability. A
protein-rich substrate (FW) was more problematic to co-digest with PM due to their similar characteristics (high nitrogen content), but good results were achieved by keeping the FW in the feeding mixture below 10%. The lipid-rich substrate (BW) helped to decrease ammonium concentrations in the reactor, but provoked VFA accumulation events, which could be solved by adjusting the BW fraction in the feeding mixture (<6%) or applying a fed-batch strategy. Acknowledgements This research was supported by the Spanish Ministry of Economy and Competitiveness, through NOVEDAR_Consolider (CSD2007-00055) and COMDIGEST (CTM2010-17196) projects, and by the Xunta de Galicia through GRC2010/37 project and the postdoctoral contract to Dr. Marta Carballa (Isidro Parga Pondal, IPP-08-37). The authors are also grateful to Laura Otero and Patricia Veiga for their experimental work.
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012.07. 109.
References Alvarez, R., Liden, G., 2008. Low temperature anaerobic digestion of mixtures of llama, cow and sheep manure for improved methane production. Biomass and Bioenergy 33, 527–533. Alvarez, J.A., Otero, L., Lema, J.M., 2010. A methodology for optimising feed composition for anaerobic co-digestion of agro-industrial wastes. Bioresource Technology 101, 1153–1158. Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143–169. Amon, B., Kryvoruchko, V., Amon, T., Zechmeister-Boltenstern, S., 2006. Methane, nitrous oxide and ammonia emissions during storage and after application of dairy cattle slurry and influence of slurry treatment. Agriculture, Ecosystems and Environment 112, 153–162. Angelidaki, I., Ellegaard, L., 2003. Codigestion of manure and Organic wastes in centralized biogas plants. Applied Biochemistry & Biotechnology 109, 95–105. Astals, S., Ariso, M., Galí, A., Mata-Alvarez, J., 2011. Co-digestion of pig manure and glycerine: Experimental and modelling study. Journal of Environmental Management 92 (4), 1091–1096. Astals, S., Nolla-Ardèvol, V., Mata-Alvarez, J., 2012. Anaerobic co-digestion of pig manure and crude glycerol at mesophilic conditions: biogas and digestate. Bioresource Technology 110, 63–70. Blume, F., Bergmann, I., Nettmann, E., Schelle, H., Rehde, G., Mundt, K., Klocke, M., 2010. Methanogenic population dynamics during semi-continuous biogas fermentation and acidification by overloading. Journal of Applied Microbiology 109 (2), 441–450. Braun, R., Wellinger, A., 2003. Potential of co-digestion. IEA Bioenergy, Task 37 – Energy from Biogas and Landfill Gas. Callaghan, F.J., Wase, D.A.J., Thayanithy, K., Forster, C.F., 2002. Continuous co-digestion of cattle slurry with fruit and vegetable wastes and chicken manure. Biomass and Bioenergy 22, 71–77. Cavaleiro, A.J., Salvador, A.F., Alves, J.I., Alves, M.M., 2009. Continuous high rate anaerobic treatment of oleic acid based wastewater is possible after a step feeding start-up. Environmental Science and Technology 43 (8), 2931–2936. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresource Technology 99, 4044–4064. Cuetos, M.J., Gómez, X., Otero, M., Morán, A., 2008. Anaerobic digestion of solid slaughterhouse waste (SHW) at laboratory scale: influence of co-digestion with the organic fraction of municipal solid waste (OFMSW). Journal of Biochemical Engineering 40, 99–106. Daims, H., Lücker, S., Wagner, M., 2006. DAIME, a novel image analysis program for microbial ecology and biofilm research. Environmental Microbiology 8, 200–213. Dasari, M.A., Kiatsimkul, P.P., Sutterlin, W.R., Suppes, G.J., 2005. Low-pressure hydrogenolysis of glycerol to propylene glycol. Applied Catalysis a-General 281, 225–231.
513
Garcia, E., Omil, F., Lema, J.M., 2003. Clean production in fish canning industries: recovery and reuse of selected wastes. Clean Technologies and Environmental Policy 5, 289–294. Guerrero, L., Omil, F., Méndez, R., Lema, J.M., 1997. Treatment of saline wastewaters from fish meal factories in an anaerobic filter under extreme ammonia concentrations. Bioresource Technology 61 (1), 69–78. Hansen, K.H., Angelidaki, I., Ahring, B.K., 1999. Improving thermophilic anaerobic digestion of swine manure. Water Research 33 (8), 1805–1810. Hartman, H., Ahring, B.K., 2006. Strategies for the anaerobic digestion of the organic fraction of municipal solid waste: an overview. Water Science and Technology 53 (8), 7–22. Hori, T., Haruta, S., Ueno, Y., Ishii, M., Igarashi, Y., 2006. Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Applied and Environmental Microbiology 72 (2), 1623–1630. Ito, T., Nakashimada, Y., Senba, K., Matsui, T., Nishio, N., 2005. Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. Journal of Bioscience and Bioengineering 100 (3), 260–265. Jeganathan, S., Von Bergen, M., Brutlach, H., Steinhoff, H.J., Mandelkow, E., 2006. Global hairpin folding of tau in solution. Biochemistry 45, 2283–2293. Karakashev, D., Schmidt, J.E., Angelidaki, I., 2008. Innovative process scheme for removal of organic matter, phosphorus and nitrogen from pig manure. Water Research 42, 4083–4090. Liu, H., Wang, J., Wang, A.J., Chen, J.A., 2011. Chemical inhibitors of methanogenesis and putative applications. Applied Microbiology and Biotechnology 89 (5), 1333–1340. Ma, J., Van Wambeke, M., Carballa, M., Verstraete, W., 2008. Improvement of the anaerobic treatment of potato processing wastewater in a UASB reactor by co-digestion with glycerol. Biotechnology Letters 30 (5), 861–867. Mata-Álvarez, J., Mace, S., Llabrés, P., 2000. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresource Technology 74, 3–16. Nielsen, H.B., Angelidaki, I., 2008. Strategies for optimizing recovery of the biogas process following ammonia inhibition. Bioresource Technology 99 (17), 7995–8001. Novak, S.M., Fiorelli, J.M., 2010. Greenhouse gases and ammonia emissions from organic mixed crop-dairy systems: a critical review of mitigation options. Agronomy for Sustainable Development 30, 215–236. Pereira, M.A., Sousa, D.Z., Mota, M., Alves, M.M., 2004. Mineralization of LCFA associated to anaerobic sludge: kinetics, transport limitations, enhancement of methanogenic activity and effect of VFA. Biotechnology and Bioengineering 88 (4), 502–511. Pullammanappallil, P.C., Chynoweth, D.P., Lyberatos, G., Svoronos, S.A., 2001. Stable performance of anaerobic digestion in the presence of a high concentration of propionic acid. Bioresource Technology 78 (2), 165–169. Robra, S., Serpa da Cruz, R., de Olveira, A.M., Almeida-Neto, J.A., Santos, J.V., 2010. Generation of biogas using crude glycerine from biodiesel production as a supplement to cattle slurry. Biomass and Bioenergy 34, 1330–1335. Standard Methods for the Examination of Water and Wastewater (APHA), 1995. American Public Health Association/American Water Works Association/ Water Environment Federation, 19th Edition Washington D.C., USA. Weiland, P., 2010. Biogas production: current state and perspectives. Applied Microbiology and Biotechnology 85, 849–860. Yazdani, S.S., Gonzalez, R., 2007. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Current Opinion Biotechnology 18, 213–219.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced methane production from pig manure anaerobic digestion using fish and biodiesel wastes as co-substrates Leticia Regueiro a, Marta Carballa a,⇑, Juan A. Álvarez b, Juan M. Lema a a b
Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Rúa Lope Gómez de Marzoa s/n, 15782 Santiago de Compostela, Spain AIMEN Technological Center, C/Relva, 27 A – Torneiros, 36410 Porriño Pontevedra, Spain
h i g h l i g h t s " Fish and biodiesel waste were used as co-substrates in pig manure anaerobic digestion. " Both co-substrates improved methane yield but caused VFA and ammonium accumulation. " Shorter HRT and FW < 10% in the feeding allow to control ammonium inhibition. " Biodiesel waste co-digestion requires feeding shares < 6% and/or fed-batch operation. " The poorer the co-digester operation, the higher the Methanosarcina/Methanosaeta ratio.
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 26 July 2012 Accepted 28 July 2012 Available online 7 August 2012 Keywords: Agroindustrial wastes Ammonia Co-digestion Methanosarcina Volatile fatty acids
a b s t r a c t Co-digestion of pig manure (PM1) with fish (FW2) and biodiesel waste (BW3) was evaluated and compared with sole PM digestion. Results indicated that co-digestion of PM with FW and/or BW is possible as long as ammonium and volatile fatty acids remained under inhibitory levels by adjusting the operating conditions, such as feed composition, organic loading rate (OLR) and hydraulic retention time (HRT). PM and FW codigestion (90:10 and 95:5, w/w4) was possible at OLR of 1–1.5 g COD/L d, resulting in biogas production rates of 0.4–0.6 L/L d and COD removal efficiencies of 65–70%. Regarding BW, good results (biogas production of 0.9 L/L d and COD elimination of 85%) were achieved with less than 5% feeding rate. Overall, operating at the same OLR, the biogas production and methane content in the co-digester was higher than in the only PM digester. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In the last years, anaerobic digestion of animal wastes has been promoted in order to avoid the uncontrolled emissions of CH4 during storage (Novak and Fiorelli, 2010). Pig manure (PM5) can be an excellent base substrate for anaerobic digestion due to its inherent buffering capacity and high content of a wide range of nutrients required for the development of anaerobic microorganisms. However, PM has a low biogas yield, around 20–30 m3/ton (Angelidaki and Ellegaard, 2003), and high ammonium concentrations (2–3 g N– ⇑ Corresponding author. Tel.: +34 881 816020; fax: +34 881 816702. E-mail addresses: [email protected] (L. Regueiro), marta. [email protected] (M. Carballa), [email protected] (J.A. Álvarez), [email protected] (J.M. Lema). 1 PM, pig manure; 2 FW, fish waste; 3 BW, biodiesel waste; 4 w/w, wet weight basis; 5 PM, pig manure; 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.109
NH4+/L). Consequently, PM is preferably co-digested with high carbon content wastes, on one hand, to improve the C/N ratio (Hartman and Ahring, 2006), and on the other hand, to increase the biogas production, essential for the plant’s economy. It has been shown that bioenergy production in farm biogas plants could be enhanced by 80–400% by using organic wastes and by-products as co-substrates (Braun and Wellinger, 2003; Weiland, 2010). Despite the wellknown reported co-digestion benefits, such as optimum humidity and C/N ratio or inhibitory substances dilution (Mata-Álvarez et al., 2000), it is not clear whether some substrates have adverse impact when they are co-digested with another waste (Callaghan et al., 2002). Therefore, it is critical to obtain an optimal mixture of the available co-substrates as well as the optimum operating conditions which allow high biogas yields without compromising the stability of the process (Alvarez et al., 2010). Fish and shellfish canning industry is an important sector in Galicia (NW of Spain), with around 65% of the total Spanish production and representing 45% of the Galician factories and 67% of the jobs (Garcia et al., 2003). This sector generates different
508
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
solid wastes with a wide range of characteristics depending on the raw material processed (tuna, mussel, sardine, mackerel, etc.). In general, fish wastes (FW6) are protein-rich substrates, although they also contain important lipids. Protein-rich materials have a fast biomethanation, but their degradation products (ammonium) can inhibit the process as well (Chen et al., 2008). Ammonium inhibition is directly related to the concentration of the undissociated form (NH3) or free ammonia (FA7), thus becoming more important at high pH levels. The inhibitory FA concentration varies depending on operational parameters such as origin of inoculum, substrate, pH and temperature (Alvarez and Liden, 2008). The reported FA inhibitory concentrations for mesophilic conditions range from 25 to 140 mg N-FA/L, whereas during the thermophilic digestion of cattle manure, higher values (around 390–700 mg N-FA/L) were tolerated after an initial acclimation period (Guerrero et al., 1997). Biodiesel fuels have recently drawn much attention given that they have various advantages over petroleum-based fuels (Ito et al., 2005). The biodiesel production is carried out by catalyzed transesterification with alcohol (usually methanol). Beside desired methylesters, this process also generates few other products, including crude glycerol or biodiesel waste (BW8), oil-pressed cakes and washing water. BW is easily separated from the aqueous phase and it is composed mainly of glycerol. In many occasions, it also contains a significant fraction of lipids due to inefficient separation systems. It is estimated that 1 kg of crude glycerol is generated per 9 kg of biodiesel produced (Dasari et al., 2005). The important increase in the biodiesel production of the last years has resulted in crude glycerol surplus that implied a dramatic 10-fold decrease in biodiesel waste prices (Yazdani and Gonzalez, 2007), and consequently, crude glycerol is often regarded as a waste stream with an associated disposal cost. This biodiesel residue is readily digestible and can be easily stored over a long period, making it an ideal co-substrate for anaerobic digestion (Ma et al., 2008), whereas its digestion as sole substrate is not viable as no nitrogen would be available for microbial populations (Robra et al., 2010). Lipid-rich materials are known to have high methane production potentials (Hansen et al., 1999), but their degradation products, the long-chain fatty acids (LCFA), are known to be inhibitors of methanogenic microorganisms (Pereira et al., 2004). Besides, operational instabilities related to sludge flotation and washout are also reported (Jeganathan et al., 2006). The aim of this work was to evaluate the use of fish waste and biodiesel waste as co-substrates to enhance the mesophilic anaerobic digestion of pig manure at laboratory scale. The effect of feeding mixture on process performance and microbial community composition was investigated. The results obtained in the co-digestion systems were compared with those obtained in a reactor treating only pig manure.
2. Methods 2.1. Wastes and inoculum PM was taken from a sewer of a 150-pig fattener and sow farm, which collects both feces and urine. PM samples were homogenized, sieved to 2 mm and stored at 4 °C until use to minimize decomposition. Different batches of PM were used throughout the experiment (>200 days) due to the impossibility of storing the total amount required. FW was delivered by a canning industry and it consisted of heads, tails, bones and viscera of tuna fish. FW was homogenized 6 7 8
FW, fish waste; FA, free ammonia; BW, biodiesel waste;
by grinding and stored at 20 °C. BW was taken from a biodiesel factory and was stored at 4 °C without pre-treatment. It contained mainly glycerol produced in the transesterification. One batch of FW and BW was sufficient for the complete study. Anaerobic granular biomass from an internal circulation reactor treating brewery wastewater was used as inoculum, with an initial in-reactor biomass concentration of about 10 g VSS/L. 2.2. Anaerobic reactors Experiments were carried out in three continuous stirred tank reactors (160 rpm, Heidolph RZR 2041), two co-digesters and one only-PM digester, constructed in methacrylate and with a working volume of 9.2 L, approximately. Reactors were operated at 35 °C by hot water recirculation. The applied feedstock mixtures were prepared every 2–3 days, diluted with tap water according to the applied organic loading rate (OLR), and stored at 4 °C prior to use. The digesters were fed manually after an equivalent volume of digester mixed liquor was removed. Temperature, pH, stirring speed and biogas production were monitored on-line. Other physico-chemical parameters (solids, chemical oxygen demand (COD), alkalinity, volatile fatty acids (VFA) and ammonium) were measured twice per week. 2.3. Operational strategy The operation of the two co-digesters was identical. They were started-up with a mixture of PM-FW-BW (84:5:11 in wet weight (w/w) basis), which was the optimum mixture obtained from linear programming (Alvarez et al., 2010), at an OLR of 0.5 g COD/L d and a hydraulic retention time (HRT) of 40 d. After the start-up (day 0–19), the operation of the co-digesters can be divided in three periods according to the feeding blend, HRT and OLR applied. In period I (days 20–59), the reactors were fed with a mixture of PM and FW (90:10, w/w) at OLR of 1 g COD/L d and HRT of 35 d. In period II (days 60–115), the percentage of FW was decreased to 5% and the OLR and HRT were increased to 1.5 g COD/L d and decreased to 30 d, respectively. In addition, three pulses of BW (5 g COD/L) were added to the reactors on days 80, 90 and 100. In the last period (period III, days 116–200), FW was replaced by BW in order to prevent ammonia inhibition, the OLR was increased to 2 g COD/L d and the HRT was reduced to 25 d. In all periods, the feeding mixture was diluted with tap water to attain the proper OLR. The start up of the only-PM digester with OLR of around 0.5 COD/L d and HRT of 20 d took 36 days. In period I (days 37–89), the OLR was increased up to 1 g COD/L d and the HRT was slightly lowered to 18 d in order to reach the target OLR, since the COD of PM was lower in this period. The OLR was further increased to 1.5 and 2 g COD/L d and the HRT was decreased to 15 and 12 d in periods II (days 90–104) and III (days 105–150), respectively. Since different batches of PM were used along the experiment (with different COD concentrations), dilution with tap water was not always required to attain the established OLR. 2.4. Analytical methods pH, COD, total solids (TS), volatile solids (VS), total suspended solids (TSS), volatile suspended solids (VSS), total Kjeldahl nitrogen (N-TKN), ammonium (N–NH4+), TA (total alkalinity) and PA (partial alkalinity) were performed following standard methods (APHA, 1995). Biogas production was measured online by Ritter milligascounters (Dr. Ing. Ritter Apparatebau GmbH, Bochum, Germany) and biogas composition was analyzed by gas chromatography (HP, 5890 Series II). VFA (acetic, propionic, i-butyric, n-butyric, i-valeric and n-valeric) were analyzed by gas
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
chromatography (HP, 5890A) equipped with a Flame Ionization Detector (HP, 7673A). Total lipids content was determined using the standard Soxhlet method (APHA, 1995) and protein concentration was calculated from the organic nitrogen content. Carbohydrates were estimated as the remaining fraction of VS after proteins and lipids were subtracted. FA concentration was calculated using the NH4+–NH3 equilibrium constant (Ka), N–NH4+ concentration (g/L), pH and temperature (T, °C), according to equations 1 and 2 (Cuetos et al., 2008):
NH3 ¼
NHþ4
ð1Þ
1 þ 10ðpKapHÞ
pKa ¼ 4 108 T3 þ 9 105 T2 0:0356T þ 10:072
ð2Þ
2.5. Fluorescent in situ hybridization (FISH) Active bacterial and archaeal populations were identified and semi-quantified by the FISH technique. Fresh biomass samples were collected from the reactors on days 70, 90, 110 and 200, disrupted and fixed with 4% paraformaldehyde solution according to the procedure described by Amann et al. (1995). Hybridization was performed at 46 °C for 90 min adjusting the formamide concentrations for each probe. The probes used were: Eub338mix (Bacteria), Arc915 (Archaea), Ms821 (Methanosarcina), Mx825 (Methanosaeta). All the details of each probe (formamide percentage, sequence and target organism) can be found in the probeBase database (http://www.microbial-ecology.net/probebase/). All probes were 50 labeled with the fluorochromes FITC and Cy3. Fluorescence signals were recorded with an acquisition system (Coolsnap, Roper Sicientific Photometrics) coupled to an Axioskop 2 epifluorescence microscope (Zeiss, Germany). DAIME program (Daims et al., 2006) was used to make the semi-quantitative counting with at least six photos taken per 20 lL of fixed sample (108–109 cells per mL). 3. Results and discussion 3.1. Wastes characterization Table 1 shows the physico-chemical characterization of the substrates (PM, FW and BW) used in this study. The three substrates had neutral pH values (between 6.9 and 7.3). PM had low TS (17 g/kg), VS (12 g/kg) and COD (30 g/kg) levels compared to FW (370 g TS/kg, 270 g VS/kg and 410 g COD/kg) and BW
Table 1 Physico-chemical characteristics (average values) of pig manure, fish waste and biodiesel waste. Parameter
Pig manurea
Fish waste
Biodiesel waste
pH Density (kg/L) TS (g/kg) VS (g/kg) CODtotal (g O2/kg) CODsoluble (g O2/kg) N-TKN (g N/kg) N–NH4+ (g N/kg) Total alkalinity (g CaCO3/kg) Proteins (g/kg) Lipids (g/kg) Carbohydrates (g/kg) COD/N ratio
6.9 ± 0.2 1.0 ± 0.0 17.3 ± 4.5 11.7 ± 5.3 29 ± 12 15.3 ± 7.1 3.3 ± 0.6 3.1 ± 0.4 7.7 ± 1.3 1.1 ± 0.2 1.5 ± 0.3 9.2 ± 3.8 8.9 ± 1.3
7.1 1.1 369 270 410 – 34 0.7 0.4 206 28 36 12.2
7.3 1.0 950 938 1390 – 0.2 0 0 1.2 77.3 922 7315
TS, total solids; VS, volatile solids; COD, chemical oxygen demand; N-TKN, total Kjeldahl nitrogen; N–NH4+, ammonium. a Standard deviations are only shown for pig manure since several batches of this substrate were used along the experiment.
509
(950 g TS/kg, 940 g VS/kg and 1390 g COD/kg), but high alkalinity (7.7 g/kg). As expected, the highest nitrogen concentration was found in FW (34 g N-TKN/kg), while it was negligible in BW. In contrast, BW had a high content of lipids (77.3 g/kg) and of easily biodegradable carbohydrates (almost 925 g/kg). In summary, PM provides moisture and alkalinity, FW provides mainly nitrogen from proteins and also lipids but in a minor proportion (mainly from fishbone fiber and cartilage) and BW provides easily degradable COD (carbohydrates) and lipids. 3.2. Continuous pig manure anaerobic co-digestion Two co-digesters were operated for 200 days at identical conditions (duplicates). The performance of the two co-digesters was very comparable, and therefore, only data from one codigester are shown in figures and tables. pH and biomass concentrations remained at 7.2–8.2 and 5–7 g VSS/L, respectively, during the whole experiment (data not shown). Fig. 1 shows the performance of one co-digester in terms of OLR and biogas production (Fig. 1A), COD concentrations in influent and effluent (Fig. 1B), VFA levels (Fig. 1C) and ammonium and FA concentrations (Fig. 1D). At the end of the start-up period (day 19), the biogas production reached 0.22 L/L d (Fig. 1A), with methane content of 50%, and the COD removal efficiency was around 61%. During this period, acetic acid was consumed, but propionic acid remained in values around 800 mg/L (Fig. 1C). This was probably due to the relatively high volumetric percentage of BW in the feeding mixture (11%), since BW is considered as rapidly biodegradable organic substrate, which generates large amounts of VFA (Astals et al., 2011). Several authors (Amon et al., 2006; Astals et al., 2011) have shown that 4–6% (w/w) of BW in the feeding is considered to be the limiting concentration to maintain a stable anaerobic digestion process. Moreover, the propionic to acetic acid ratio at the end of the start-up was about 3.8 and it has been reported that a propionic to acetic acid ratio greater than 1.4 is a hint of immediate digester failure (Pullammanappallil et al., 2001). Thus, in period I, BW was eliminated from the feeding mixture. In the first days of period I (days 20–30), when the co-digester was fed with a mixture of PM and FW (90:10, w/w), the VFA content decreased to less than 0.2 g/L, despite the OLR doubling i.e. from 0.5 to 1 g COD/L d. This fact suggests that the elimination of BW from the feeding mixture had a satisfactory effect on process stability. Consequently, the biogas production and the COD removal increased to 0.43 L/L d and 70%, respectively. But from day 30 on, ammonium concentrations started rising (Fig. 1D) as a result of the digestion of the proteins present in FW and the high ammonium levels in PM (3 g N–NH4+/kg, Table 1). Although no accumulation of VFA was observed (Fig. 1C), total COD concentrations in the effluent increased from 9 to 11.5 g/L (Fig. 1B) and biogas production decreased to 0.25 L/L d (Fig. 1A) by the end of this period. To attenuate the ammonium accumulation, the percentage of FW in the feeding mixture and the HRT were decreased in period II in order to promote ammonium wash out. As expected, ammonium levels decreased during the first days of period II, but from day 80 on, they increased again (Fig. 1D). The change in the PM stock (with higher ammonium concentrations) combined with the higher OLR applied in this period (1.5 g COD/L d, Table 2) provoked this increase in the ammonium levels in the reactors. In period II, three pulses of BW (5 g COD/L) were performed on days 80, 90 and 100, following the strategy proposed by Cavaleiro et al. (2009) to achieve efficient methane yields from lipid-rich substrates. This strategy consists of the combination of lipid-rich substrate feeding periods (LCFA accumulation) with non-feeding periods (degradation of accumulated LCFA). These authors
510
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Fig. 1. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (BW pulses in period II were not considered in influent COD (CODin)) (B), volatile fatty acids (VFA) concentration (C) and ammonium and free ammonia (FA) concentrations (D) in one co-digester. Arrows in Fig. 1A indicate BW pulses of 5 g COD/L on days 80, 90 and 100.
Table 2 Operational parameters of the lab-scale anaerobic co-digester and only-pig manure digester during the different operational periods (values shown corresponded to steady-state conditions of each period).
a
Type reactor
Period
PM-FW-BW (% w/w)a
Duration (days)
OLR (g COD/L d)
HRT (d)
pH
CH4 (%)
COD removal (%)
Biogas (L/L d)
Co-digester
Start-up Period I Period II Period III
84–5-11 90–10-0 95–5-0 95–0-5
0–19 20–59 60–115 116–200
0.5 1.0 1.5 2.0
40 35 30 25
7.4 ± 0.2 7.6 ± 0.3 8.0 ± 0.2 7.7 ± 0.2
50 ± 2 57 ± 3 59 ± 2 60 ± 3
60.8 ± 0.3 69.6 ± 1.2 65.7 ± 0.8 78.5 ± 0.7
0.22 ± 0.01 0.43 ± 0.04 0.59 ± 0.05 0.91 ± 0.06
Digester
Start-up Period I Period II Period III
0–36 37–89 90–104 105–150
0.6 1.0 1.5 2.0
20 18 15 10
7.6 ± 0.2 7.9 ± 0.2 8.0 ± 0.2 8.1 ± 0.1
56 ± 4 52 ± 7 52 ± 5 55 ± 6
40.2 ± 0.4 37.5 ± 0.9 40.1 ± 2.0 52.6 ± 3.7
0.15 ± 0.02 0.25 ± 0.03 0.40 ± 0.01 0.67 ± 0.10
PM
w/w: wet weight.
reported that at least two cycles of accumulation-degradation of LCFA were necessary before continuous operation The positive effect of BW pulses on biogas production was clear (Fig. 1A), but
the % of pulse COD converted into methane in relation to the maximum theoretical methane production (taking into account 0.35 m3 CH4/kg CODconverted) decreased with each consecutive
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
pulse (52.1%, 26.8% and 24.2%), probably as a result of the increase in VFA levels after the first pulse addition (Fig. 1C). The major components of the VFAs were acetic and propionic acid, which reached values up to 2.5 g/L and 2.0 g/L, respectively. The increased VFA levels also resulted in higher effluent COD concentrations (Fig. 1B), and consequently, the COD removal efficiency decreased from 70% (before pulses) to 66% (after pulses). By the end of period II, VFA and the COD levels lowered, but did not reach the original values (Fig. 1B and C), indicating that organic compounds related to BW, such as alcohols or LCFA, were accumulated into the co-digesters. In contrast, the biogas production was not inhibited, suggesting that the degradation of PM:FW mixture was not affected and just the COD from pulses was accumulated. By the end of period II, the ammonium levels were still high (2 g N–NH4+/L), which combined with high pH values (around 7.8, data not shown) resulted in FA levels around 260 mg N–NH3/L, which might be inhibitory for anaerobic communities (Chen et al., 2008). Furthermore, FA levels were much more dependent on pH variations than on the ammonium concentration, because between days 106 and 112, FA increased from 125 to 260 mg N–NH3/L, when the pH varied from 7.8 to 8.1, despite the decrease of the ammonium levels from 2.2 to 2 g N–NH4+/L (Fig. 1D). Consequently, FW was replaced by BW in period III. In this case, the percentage of BW in the mixture (5%) did not exceed the 6% that Amon et al. (2006) set as maximum for a good performance. Besides, HRT was decreased to 25 d in order to promote ammonium washout. These changes led to a drop in ammonium and FA concentrations, which stabilized at 1300 mg N–NH4+/L and 100 mg N–NH3/L, respectively, by the end of the experiment (Fig. 1D). In period III, biogas production increased as a consequence of the higher OLR applied (Fig. 1A). However, from day 130, it went down due to the accumulation of propionic acid (Fig. 1C), resulting on 0.3 L/L d on day 150. To surpass this inhibition, the co-digesters were not fed between days 151 and 160 and propionic acid was consumed giving rise to a recovery in biogas production rates. After this event, the co-digesters operated stably for 40 days, with biogas production rates of 0.9 L/L d and COD removal efficiencies of almost 80%, which emphasizes the high biodegradability of BW. The microbiology of the co-digesters in the last two operational periods was studied with the FISH technique (Supplementary material (SM), Fig. SM 1). A rough estimation of the relative shares of each main population indicated that half of the active population was Archaea and the other half was Bacteria. This ratio did not vary, even when the feeding mixture changed as happened between days 100 and 120. The relative abundance of archaeal populations (Methanosaeta and Methanosarcina) present was correlated with the co-digester performance, particularly with VFA concentrations. Before the pulses (day 70), the predominant archaeal population was Methanosaeta (Fig. SM-1A), demonstrated by the presence of the characteristic shape of this population in tubular sheath. Methanosaeta are well known as the most abundant acetoclastic methanogens in bioreactors with low VFA and ammonium levels (Karakashev et al., 2008), as occurred before the pulses. After the three pulses (day 90), this population disappeared almost completely and the niche was filled by Methanosarcina, which displayed the individual coccoid cells shape forming aggregates similar to a bunch of grapes (Fig. SM-1B). Methanosarcina is able to use both the acetoclastic and the hydrogenotrophic pathways, thus being more tolerant to methanogenesis inhibitors compared to Methanosaeta (Liu et al., 2011). Its presence in anaerobic reactors is often associated with the deterioration of reactor performance (Blume et al., 2010; Hori et al., 2006). Consequently, the appearance of this archaeal population is probably related to the VFA accumulation that occurred between days 85 and 110 (Fig. 1C). On day 120 (Fig. SM-1C), when
511
the co-digester was recovering from inhibition (VFA concentration was decreasing), the two methanogenic populations coexisted in the reactor. At the end of the experiment (day 200), when the co-digester showed a good stable performance, the methanogenic population was again mainly Methanosaeta (Fig. SM-1D). These variations in Methanosaeta and Methanosarcina populations were semi-quantified by using the probes Mx825 and Ms821, respectively. Methanosaeta fractions (with respect to total archaeal population) were 95%, 0%, 45% and almost 100% on days 70, 90, 110 and 200, respectively, while those of Methanosarcina were 0%, 100%, 40% and 0%, respectively. 3.3. Continuous pig manure anaerobic digestion pH and biomass concentrations remained at 7.4–8.3 and 5–8 g VSS/L, respectively, during the whole experiment (data not shown). Fig. 2 shows the results of the operation of the reactor treating PM as a sole substrate. During the start-up (day 0–36), biogas production stayed at around 0.15 L/L d (Fig. 2A) with methane content of 56%, leading to average COD removal efficiencies of 40% (Fig. 2B). VFA concentrations were negligible (Fig. 2C) and ammonium levels remained below 1.2 g N–NH4+/L (Fig. 2D). The increase in the OLR from 0.6 to 1 g COD/Ld in period I derived in greater biogas production rates (0.25 L/L d, methane content of 52%), but the COD removal efficiencies remained at lower values (around 38%). The accumulation of ammonium observed at the end of the start-up continued and stabilized at around 3 g N–NH4+/L (Fig. 2D). The latter combined with the accumulation of VFA between days 65 and 80 (Fig. 2C) provoked a drop on the biogas production, and consequently, on the COD removal efficiencies (Fig. 2B). Although the reactor recovered quite quickly from VFA accumulation, ammonium concentrations were still high (Fig. 2D), and therefore, the HRT was decreased to 15 days in period II. This modification combined with a higher COD content in PM yielded an increase in the OLR to 1.5 g COD/L d. Consequently, biogas production and COD removal efficiencies increased to 0.40 L/L d and 40%, respectively. Since ammonium levels did not decrease, the HRT was further lowered to 10 days on day 115 (period III). Despite the fluctuations in the OLR (1.6–2.2 g COD/L d, Fig. 2A) caused by variations in COD content of PM, biogas production remained quite constant at 0.7 L/L d (Fig. 2A). By the end of the experiment, VFA concentrations were negligible (Fig. 2C), but ammonium and FA stayed at around 2.5 g N–NH4+/L and 250 mg N–NH3/L, respectively (Fig. 2D). 3.4. Improved pig manure anaerobic digestion by using FW and BW Table 2 shows a comparison of the results obtained in the co-digester and in the only-PM digester at steady-state conditions during each operational period. Operating at the same OLR, the biogas production and methane content in the co-digester were higher than in the only-PM digester. More specifically, biogas production was improved by 47%, 72%, 48% and 36% at OLR of 0.5, 1, 1.5 and 2 g COD/L d, respectively. Moreover, the COD converted into methane was much higher in the co-digestion process, reaching values near to 79% (period III), while in the digestion process the maximum value was 53% (period III). An additional advantage of the co-digestion process was the ammonium and FA control in the reactors. The highest values achieved in the co-digester (2 g N–NH4+/L and 250 mg N–NH3/L) were around 1.5-fold lower than those in the only-PM digester (3.2 g N–NH4+/L and 320 mg N–NH3/L). Although anaerobic microorganisms can adapt to relatively high FA levels, some studies suggested that microorganisms adapted to the anaerobic digestion of PM were inhibited at FA concentrations around 0.7–1.1 g N–NH3/L (Nielsen and Angelidaki, 2008).
512
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Fig. 2. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (B), volatile fatty acids (VFA) concentration (C) and ammonium and free ammonia (FA) concentrations (D) in the pig manure anaerobic reactor.
The main problems associated to FW and BW were ammonium and VFA accumulation, respectively. The first can be surpassed by working at low OLR as in period II (around 1.5 g COD/L d) and/or at shorter HRT to promote ammonium washout, and the second one can be solved by adapting the feeding strategy of BW. In this study, two alternatives were evaluated: pulses in period II and continuous operation in period III. Not good results were obtained with the pulses, probably due to their frequency and COD concentration, while the continuous operation with BW percentages below 6% in the feeding mixture derived a stable performance with good biogas production (0.91 L/L d), which is consistent with results obtained by other authors (Astals et al., 2012; Robra et al., 2010). 4. Conclusions Anaerobic co-digestion of PM with FW or BW evidenced an upgrade with respect to sole PM digestion, not only in terms of increased biogas production, but also on process stability. A
protein-rich substrate (FW) was more problematic to co-digest with PM due to their similar characteristics (high nitrogen content), but good results were achieved by keeping the FW in the feeding mixture below 10%. The lipid-rich substrate (BW) helped to decrease ammonium concentrations in the reactor, but provoked VFA accumulation events, which could be solved by adjusting the BW fraction in the feeding mixture (<6%) or applying a fed-batch strategy. Acknowledgements This research was supported by the Spanish Ministry of Economy and Competitiveness, through NOVEDAR_Consolider (CSD2007-00055) and COMDIGEST (CTM2010-17196) projects, and by the Xunta de Galicia through GRC2010/37 project and the postdoctoral contract to Dr. Marta Carballa (Isidro Parga Pondal, IPP-08-37). The authors are also grateful to Laura Otero and Patricia Veiga for their experimental work.
L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012.07. 109.
References Alvarez, R., Liden, G., 2008. Low temperature anaerobic digestion of mixtures of llama, cow and sheep manure for improved methane production. Biomass and Bioenergy 33, 527–533. Alvarez, J.A., Otero, L., Lema, J.M., 2010. A methodology for optimising feed composition for anaerobic co-digestion of agro-industrial wastes. Bioresource Technology 101, 1153–1158. Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143–169. Amon, B., Kryvoruchko, V., Amon, T., Zechmeister-Boltenstern, S., 2006. Methane, nitrous oxide and ammonia emissions during storage and after application of dairy cattle slurry and influence of slurry treatment. Agriculture, Ecosystems and Environment 112, 153–162. Angelidaki, I., Ellegaard, L., 2003. Codigestion of manure and Organic wastes in centralized biogas plants. Applied Biochemistry & Biotechnology 109, 95–105. Astals, S., Ariso, M., Galí, A., Mata-Alvarez, J., 2011. Co-digestion of pig manure and glycerine: Experimental and modelling study. Journal of Environmental Management 92 (4), 1091–1096. Astals, S., Nolla-Ardèvol, V., Mata-Alvarez, J., 2012. Anaerobic co-digestion of pig manure and crude glycerol at mesophilic conditions: biogas and digestate. Bioresource Technology 110, 63–70. Blume, F., Bergmann, I., Nettmann, E., Schelle, H., Rehde, G., Mundt, K., Klocke, M., 2010. Methanogenic population dynamics during semi-continuous biogas fermentation and acidification by overloading. Journal of Applied Microbiology 109 (2), 441–450. Braun, R., Wellinger, A., 2003. Potential of co-digestion. IEA Bioenergy, Task 37 – Energy from Biogas and Landfill Gas. Callaghan, F.J., Wase, D.A.J., Thayanithy, K., Forster, C.F., 2002. Continuous co-digestion of cattle slurry with fruit and vegetable wastes and chicken manure. Biomass and Bioenergy 22, 71–77. Cavaleiro, A.J., Salvador, A.F., Alves, J.I., Alves, M.M., 2009. Continuous high rate anaerobic treatment of oleic acid based wastewater is possible after a step feeding start-up. Environmental Science and Technology 43 (8), 2931–2936. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresource Technology 99, 4044–4064. Cuetos, M.J., Gómez, X., Otero, M., Morán, A., 2008. Anaerobic digestion of solid slaughterhouse waste (SHW) at laboratory scale: influence of co-digestion with the organic fraction of municipal solid waste (OFMSW). Journal of Biochemical Engineering 40, 99–106. Daims, H., Lücker, S., Wagner, M., 2006. DAIME, a novel image analysis program for microbial ecology and biofilm research. Environmental Microbiology 8, 200–213. Dasari, M.A., Kiatsimkul, P.P., Sutterlin, W.R., Suppes, G.J., 2005. Low-pressure hydrogenolysis of glycerol to propylene glycol. Applied Catalysis a-General 281, 225–231.
513
Garcia, E., Omil, F., Lema, J.M., 2003. Clean production in fish canning industries: recovery and reuse of selected wastes. Clean Technologies and Environmental Policy 5, 289–294. Guerrero, L., Omil, F., Méndez, R., Lema, J.M., 1997. Treatment of saline wastewaters from fish meal factories in an anaerobic filter under extreme ammonia concentrations. Bioresource Technology 61 (1), 69–78. Hansen, K.H., Angelidaki, I., Ahring, B.K., 1999. Improving thermophilic anaerobic digestion of swine manure. Water Research 33 (8), 1805–1810. Hartman, H., Ahring, B.K., 2006. Strategies for the anaerobic digestion of the organic fraction of municipal solid waste: an overview. Water Science and Technology 53 (8), 7–22. Hori, T., Haruta, S., Ueno, Y., Ishii, M., Igarashi, Y., 2006. Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Applied and Environmental Microbiology 72 (2), 1623–1630. Ito, T., Nakashimada, Y., Senba, K., Matsui, T., Nishio, N., 2005. Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. Journal of Bioscience and Bioengineering 100 (3), 260–265. Jeganathan, S., Von Bergen, M., Brutlach, H., Steinhoff, H.J., Mandelkow, E., 2006. Global hairpin folding of tau in solution. Biochemistry 45, 2283–2293. Karakashev, D., Schmidt, J.E., Angelidaki, I., 2008. Innovative process scheme for removal of organic matter, phosphorus and nitrogen from pig manure. Water Research 42, 4083–4090. Liu, H., Wang, J., Wang, A.J., Chen, J.A., 2011. Chemical inhibitors of methanogenesis and putative applications. Applied Microbiology and Biotechnology 89 (5), 1333–1340. Ma, J., Van Wambeke, M., Carballa, M., Verstraete, W., 2008. Improvement of the anaerobic treatment of potato processing wastewater in a UASB reactor by co-digestion with glycerol. Biotechnology Letters 30 (5), 861–867. Mata-Álvarez, J., Mace, S., Llabrés, P., 2000. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresource Technology 74, 3–16. Nielsen, H.B., Angelidaki, I., 2008. Strategies for optimizing recovery of the biogas process following ammonia inhibition. Bioresource Technology 99 (17), 7995–8001. Novak, S.M., Fiorelli, J.M., 2010. Greenhouse gases and ammonia emissions from organic mixed crop-dairy systems: a critical review of mitigation options. Agronomy for Sustainable Development 30, 215–236. Pereira, M.A., Sousa, D.Z., Mota, M., Alves, M.M., 2004. Mineralization of LCFA associated to anaerobic sludge: kinetics, transport limitations, enhancement of methanogenic activity and effect of VFA. Biotechnology and Bioengineering 88 (4), 502–511. Pullammanappallil, P.C., Chynoweth, D.P., Lyberatos, G., Svoronos, S.A., 2001. Stable performance of anaerobic digestion in the presence of a high concentration of propionic acid. Bioresource Technology 78 (2), 165–169. Robra, S., Serpa da Cruz, R., de Olveira, A.M., Almeida-Neto, J.A., Santos, J.V., 2010. Generation of biogas using crude glycerine from biodiesel production as a supplement to cattle slurry. Biomass and Bioenergy 34, 1330–1335. Standard Methods for the Examination of Water and Wastewater (APHA), 1995. American Public Health Association/American Water Works Association/ Water Environment Federation, 19th Edition Washington D.C., USA. Weiland, P., 2010. Biogas production: current state and perspectives. Applied Microbiology and Biotechnology 85, 849–860. Yazdani, S.S., Gonzalez, R., 2007. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Current Opinion Biotechnology 18, 213–219.