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Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process
Renewable Energy xxx (2016) 1e8
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Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process I. Zarkadas a, *, G. Dontis a, G. Pilidis b, D.A. Sarigiannis a a b
Aristotle University of Thessaloniki, Department of Chemical Engineering, Environmental Engineering Laboratory, 54124, Thessaloniki, Greece University of Ioannina, Department of Biological Application and Technology, University Campus, 45110, Ioannina, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2015 Received in revised form 11 February 2016 Accepted 15 March 2016 Available online xxx
Mink farming is a well-established economic activity with a significant environmental footprint. In this work mink farming derived organic waste was assessed, for the first time, as substrate to anaerobic digestion plants. The substrates assessed were; (a)fresh mink manures, (b)weathered mink manures, (c) waste feed and (d)mink derived meat and bone meal. Substrates with in inoculum to substrate ratio of 2 offered specific methane productions ranging between 368 and 591 mLCH4/gVSadded corresponding to 67.4 and 91.1% of their theoretical methane potential. In the second phase of the experiments three organic loading rates and three inoculum to substrate ratios were assessed. Substrate/inoculum ratios, in batch mode, lower than 1 seem to affect negatively the process, due to slow hydrolysis and acetogenesis of the macromolecules. In addition, initial organic loading rates of up to 50 gVS/L can be applied in batch systems when manure is utilized as substrate. In contrast to this, when mink derived byproducts are used the same loading rate will result into an irreversible process inhibition due to the accumulation of intermediate products. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic digestion Mink waste Bio methane production Farming waste Meat and bone meal Biogas production
1. Introduction European fur production is a dynamic and well established industry with long tradition in the production of quality products and significant exporting experience and capabilities. The European Union is the main exporter of pelts worldwide accounting for the 64% of the total production with the States of Denmark, Netherlands, Finland and Greece being the main producers [1]. Other significant fur producers are the United States of America and China, with China being the main exporter of fur derived commodities and clothing. The specie farmed for its fur is the American Mink (Neovison vison/Mustela vison) which is a carnivore, semiaquatic mammal [2], with the full grown males reaching the 45 cm in length, 20 cm in height and the 2 kg in weight. The diet of the farmed animals is composed of low cost protein and fat rich byproducts, including slaughterhouse and aquaculture wastes [3]. The wastes generated from mink breading facilities includes manure and waste feed. Both are collected under the animal cages in small piles of up to 50 cm in height. The waste management
* Corresponding author. E-mail addresses: [email protected] (I. Zarkadas), [email protected] (G. Dontis), [email protected] (G. Pilidis), [email protected] (D.A. Sarigiannis).
options for this waste stream are constrained by the high solids, organics and nitrogen content that are significantly hampering the abilities of aerobic biological processes to treat or valorize them. Even so, composting is widely employed, due to low cost [4]. Mink farmers in order to stimulate the application of the generated compost offer it free of charge. However, acceptance by local farmers and application rates to the fields are disappointing; bad odors one of the main reasons for this rejection. Manure stockpiling and composting on the other hand can promote nitrogen and phosphorus leachate and runoff formations leading to algae blooms and eutrophication of surface waters while, contamination of underground waters with fecal microorganisms is not uncommon. After pelting dead mink bodies are either sent for incineration or rendering where they are converted into bone and meat meal (BMM). The EC Animal By-product Regulation enforces the pretreatment of the dead minks through crushing, milling and cooking at 133 C and at least 3 bars of pressure for 20 min. The high protein meal after the process is used as soil amendment (fertilizer) or as alternative fuel. Unfortunately, this market is undeveloped and the ready product is stockpiled. Bone and meat meal on the other hand can become a hazardous commodity during both storage as well as transportation due to its tendency to self-heating and igniting without the requirement for an external source of heat or fire [5].
http://dx.doi.org/10.1016/j.renene.2016.03.056 0960-1481/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: I. Zarkadas, et al., Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process, Renewable Energy (2016), http://dx.doi.org/10.1016/j.renene.2016.03.056
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Abbreviations TOC TS AD HRT FMM WMM WF BMM
Total Organic Carbon Total Solids Anaerobic Digestion Process Hydraulic Retention Time Fresh Mink Manure Weathered Mink Manure Waste Mink Feed Mink Derived Bone and Meat Meal
A waste management option which can be employed for the valorization of fur farming wastes is anaerobic digestion (AD). AD is a biomass bio-conversion process disengaged from weather conditions which offers the advantages of self-sustainability, income generation and waste valorization with limited material requirements. Anaerobic digestion of manures and plant waste material offers the opportunity for recovery of both biogas (a methane rich gas) and hygienized and nearly odorless stabilized digestate which can be applied onto agricultural land as an organic fertilizer. Anaerobic digestion has been assessed and applied to a plethora of different substrates/wastes/low value or negative value by products including animal manures [6], industrial wastes and wastewaters, municipal solid wastes, energy crops [7] and mixtures of different substrates in co-digestion schemes [8,9]. The substrates evaluated in this work were fresh and weathered mink manures (FMM, WMM respectively), waste mink feed (WF) and bone and meat meal (BMM) that is being generated by the mink carcasses after pelting. The scientific community has shown very limited interest for the management of the mink farming waste and byproducts. This can be due either to the absence of these operations in most of the developed world or due to the negative publicity that these operations have as an effect of the number of animals sacrificed so that their furs become high value human clothing and accessories. This is the first time that the mink farming waste is assessed as candidate substrate to anaerobic digestion process. 2. Material and methods 2.1. Substrates and inoculum The substrates selected included all important waste streams generated during the life cycle of minks. Unfortunately seasonality is a significant problem as most of the wastes are generated between May and November. During winter and early spring only low volumes of manure and waste feed are produced by the males and the breading females. The substrates were assessed in batch vials under mesophilic conditions mainly due to the known problems related to the inhibition of the process by elevated concentrations of unionized ammonia. This inhibitory process is driven by the bio-conversion, of protein into ammonia and it is assisted by the high temperature and pH experienced in thermophilic systems. Inoculum was collected from two 6 L laboratory scale CST reactors treating mixtures of cattle manure and food waste and operating under an OLR of 3.1 kgVSm3R-d and a HRT of 28 days. Prior to the preparation of the batches the inoculum was degassed for 1 week. Mink manure was collected from a medium size farm housing approximately 20.000 minks and located in the area of Kastoria. Fresh mink manure was considered the manure located at the upper part of the pile that kept its as excreted shape; weathered manure was considered the one located inside the piles, which had
lost its shape and its physical appearance had been altered into a thick paste. The smell of both substrates were strong and unpleasant. Waste feed was collected from the same farm and composed by the uneaten feed provided on top of each mink cage. BMM was collected from a rendering operation which is responsible for the disposal, during the pelting season, of the mink bodies and operating according to the EC Animal Byproduct Regulation. In this facility waste fat is recovered and sent for biodiesel production, while BMM is sold as fertilizer or as renewable fuel. 2.2. Batch preparation During the preparation of the batches, the vials were filled with the inoculum, the substrates and distilled water was used as filler to the active volume of 120 mL. Finally the oxygen removed through nitrogen bubbling and the batches were placed within a tempera ture controlled cabinet at 36 ± 1 C for 30 days. The same steps were used for the preparation of the batches utilized for the assessment of the different I/S ratios except that instead of water, 0.02 M potassium phosphate buffer (pH 7.2) was used for inoculum dilution. The volumetric gas production assessed daily, pH and VFA concentrations analyzed every 3e5 days and all experiments took place in triplicate. In this work three inoculum to substrate ratios 2,1, 0.5 (initial OLR of 12.5 kgVSm3) as well as three organic loading rates have been assessed. The data related to the batch preparation are presented in Table 1. To date there is a lack of standard methodologies for the application of bio methane potential test [10]. The assessment of the effect of the inoculum to substrate ratio in the digestion of different substrates is done through stepwise increase of the organic loading [11,12]. Although this is technically correct, in many cases, especially when high solid substrates are assessed, it offers data related to the tolerance of the process in extreme loading rates not applicable in real life digestion systems. In this work the idea of keeping constant the OLR and diluting, with a pH buffer, the inoculum added into the systems has also been assessed. Such an approach has been used in the past by Hashimoto [13], and et al. [14] when examining the biodegradability of straw, Brule silage and green clippings. The dilution of the inoculum is affecting a number of operational parameters, including the number of the available cells and the concentration of trace elements [14]. At the same time the presence of an inhibitor might be revealed through this stepwise process. Successful digestion demonstrates the acceptability of the substrate by the process. 2.3. Analytical methods 2.3.1. Volatile fatty acids, TOC, TKN, pH, soluble COD & biogas quality, ammonia For VFA analysis samples were centrifuged to 9000 RPM for 10 min and the supernatant were analyzed with a Shimadzu
Table 1 Composition of the experimental series. Experimental series
OLR 12.5 kgVS/m3
OLR 25 kgVS/m3
OLR 50 kgVS/m3
FMM(g FM) WMM(g FM) WF (g FM) BMM(g FM)
4.08 5.16 4.10 1.84 I/S 2 3.01 4.08 5.16 4.10 1.84
8.17 10.33 8.21 3.69 I/S 1 1.50 4.08 5.16 4.10 1.84
16.34 20.66 16.42 7.39 I/S 0.5 0.75 4.08 5.16 4.10 1.84
Inoculum (gVS) FMM(g FM) WMM(g FM) WF(g FM) BMM(g FM)
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I. Zarkadas et al. / Renewable Energy xxx (2016) 1e8
GC17A Gas Chromatographer with a flame ionization detector (GC-FID). For the separation of VFA's a Nukol 15 m 0.53 mm column was used. The volume of the generated gas was measured according to the liquid displacement principle as described by Zarkadas and Pilidis [15] with the application of inverted cylinders. Sodium hydroxide 2N was used for carbon dioxide scrubbing with the concentration of methane to be validated with a Shimadzu GC 2014 coupled to a thermal conductivity detector. A CARBOXEN 1000 60/80 Column of 15 ft 1/8in was used. TOC analysis conducted with a Shimadzu TOC-VCPH carbon analyzer coupled to a solid state combustion unit- SSM-5000A, total organic carbon concentrations were obtained by deducing the inorganic carbon from the total carbon readings. Total Solids and Volatile solids analyzed as described by Standard Methods, (APHA 1989). Total Kjeldahl Nitrogen was analyzed with a HACH Digesdahl Digestion Apparatus and the method 8075. pH analysis was conducted with a Hanna pH 211 microprocessor pH meter. For soluble COD analysis the samples were diluted with distilled water addition in a ratio of 1/1, followed by manual mixing and the samples filtered through 0.45 mm pore size filters with the utilization of vacuum vials and a hand pump. The analysis was conducted with the application of HACH COD cuvettes and a DR2000 direct reading photometer. The ammonia concentration of the manure was analyzed using a Hanna HI 84185 coupled to an ammonia selective electrode. The theoretical methane production calculated according to[16] and the application of the following conversion formula: CH4m3/kgVS ¼ (0.496X) þ (1.014Y) þ (0.415Z)
Where: X ¼ % Protein fraction of VS Y ¼ % Fat fraction of VS Z ¼ % Carbohydrates fraction of VS
2.3.2. Statistical analysis The significance of differences in methane yield and content determined by applying single factor Analysis of Variance (ANOVA) in MATLAB R2012b. Statistical significance was established at a PValue of less than 0.05. 3. Results and discussion In this study four types of mink farming waste, were assessed as substrates to mesophilic anaerobic digestion. Their characteristics are given in Table 2. The substrates are in the solid state with TS higher than 33%. The solids are composed mainly of organic matter with VS levels higher than 83%. They have significant nitrogen content with TKN concentrations ranging between 14 and 93 g/Kg, while the pH of the manure is alkaline. This is in contrast to that of
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the WF, which is acidic and that of BMM, which is neutral. Finally the theoretical methane production of the substrates range between 545 and 705 mLCH4/gVSadded. The experiments were divided into two stages. In the first part the bio methane potential of the different substrates were investigated in an inoculum to substrate ratio of 2 and an OLR of 12.5 kgVS/m3, as these operational parameters have been shown by other researches [17,18] as appropriate for: a) providing the required number of microorganisms that will initiate a robust process b) minimizing the likelihood of overloading the process by intermediate products accumulation, including the volatile fatty acids, and c) reducing latency and occurrence of an initial lag phase. In the second part of the experiment, two additional inoculum to substrate ratios were examined with another two organic loading rates. Low initial inoculum loadings were examined in order to identify and assess the compatibility and acceptance of the substrates by the anaerobic digestion microorganisms. 3.1. Biomethane potential Fig. 1 shows the cumulative and daily methane production of the different substrates. Methane production started rapidly without the presence of a lag phase. The highest production was observed by BMM with 591 ± 38 mL/gVSadded, a value corresponding to the 83.7% of the theoretical methane production for this substrate. Bone and meat meal sourced from different facilities have been assessed in the past in relation to their bio methane potentials and the possibility of these products to be used as substrates to anaerobic digestion plants. Most frequently BMM available on the market is produced from waste generated during the slaughtering of farmed animals. This waste together with blood, feather and bone meals are the byproducts of slaughtering and dressing animals ready for human consumption. In all cases the soft edible tissues are removed and sold for human consumption, while the BMM is produced from the offal, condemned meat and the bones. Mink BMM includes also soft tissue as it is not possible for the mink meat to be utilized further and it must be disposed or valorized through processes alternative to human or pet animal food/feed manufacturing. The bio methane potential of the BMM assessed in the present study was significantly higher compared to the values found in the relevant literature [19,20]. There specific methane production fluctuates between 225 and 390 mLgVSadded. An explanation to the significantly higher production of the mink derived BMM might be the difference in the fat and protein content of the products assessed. In the marketable bone and meat meal, fat content fluctuates between 12 and 14% in agreement with the literature value of 11.9% reported by Hejnfelt and Angelidaki [19].
Table 2 Characterization of the substrates utilized (N/A-not analyzed).
Total solids (%) Volatile solids (%TS) Total Kjeldahl nitrogen g/kg NH3-N (g/kg) Total organic carbon (%TS) Total fat (%TS) pH Soluble COD (g/kg) Theoretical CH4 production mLCH4gVSadded
Inoc.
FMM
WMM
WF
BMM
5.12 80.7 1.69 N/A 30.4 0.26 7.81 N/A e
41.8 88.3 17.9 5.74 43.8 7.52 9.12 14.5 545.5
33.9 86.1 14.4 4.96 41.6 5.19 8.53 25.9 543.0
39.7 92.5 31.2 N/A 44.6 16.9 5.90 N/A 601.0
97.4 83.8 93.1 N/A 47.2 21.4 7.12 N/A 705.9
Please cite this article in press as: I. Zarkadas, et al., Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process, Renewable Energy (2016), http://dx.doi.org/10.1016/j.renene.2016.03.056
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Fig. 1. (A) Daily specific methane production, (B) cumulative methane production versus total VFA development.
straw, which is provided to the animal as bedding material. Cellulose and especially its crystalline form is one of the most difficult natural macromolecules for degradation under anaerobic conditions [25]. During sampling and preparation of the experiments in the FMM long straws were visible, unlike what happened in the case of WMM where only short straw pieces were visible; the latter were much softer and easily crashed by hand. Furthermore, mink manure is rich in ammonia, a chemical known to be used during pretreatment of straw or cellulose rich substrates prior to anaerobic digestion or bioethanol production per se [26,27]. Ammonia application and pretreatment promotes hydrolysis and release of simple cellulose sugars deprived of the negative effects of the lignin which is not converted anaerobically. Mink manure is formed by the animals in piles, which are removed twice per year. The in-pile retention time of the manure in the presence of ammonia can promote straw hydrolysis, and in turn improve conversion efficiency and bio-methane production. Another plausible mechanism leading to enhanced hydrolysis could be micro aeration [28]. The anoxic conditions formed promote microorganisms that increase the solution of the substrate. A third possible mechanism could be the weathering of an inhibitor present in the FMM but not in the WMM is also possible, however no such inhibitor was identified in the present study. The daily methane production was also higher for the WMM compared to the FMM with the daily productivity, during the exponential phase, averaging at 52.9 and 60.6 mLCH4gVSadded for the FMM and the WMM respectively. Thus WMM resulted in a 13% higher bio-methane production than FMM. 3.3. Waste mink feed
In contrast to this the fat content of BMM in our study was higher than 20%. Considering that the bio methane potential of fat is roughly twice that of both carbohydrates and proteins, the increased values achieved could be the effect of the high fat content in the substrate. Furthermore the bone and meat meal used in other literature studies had at least 10% more protein, rendering the formation of ammonia and the resulting process inhibition a formidable problem. Daily methane production during the exponential phase for the BMM was found to mount to 63.6 mL/gVSadded presenting two production peaks on days 4 and 8, probably due to the different bio-conversion rates of the substrate components. 3.2. Fresh and weathered mink manure The bio methane potential of the mink manure was 368 and 428 mLCH4gVSadded for the fresh and weathered manures respectively. Mink as a monogastric carnivore requires feed rich in protein and fat while its digestion track efficiency fluctuates between 68.9 and 74.3% with the fat being readily digested with up to 90% [2]. The bio methane potential of mink manure is significantly higher when compared to common anaerobic digestion substrates including the manure generated by pigs, cattle or poultry; the latter rarely exceed the specific methane production of 300 mLCH4gVSadded [21,22]. Thus, the bio methane potential of mink manure is at par with high quality food waste [23] and slaughterhouse waste [24]. The bio methane potential of WMM was 14% higher than that of FMM. This is counterintuitive, since in general weathering affects negatively the quality of the substrates as part of it is lost as carbon dioxide to the atmosphere. In the case of mink manure, materials with lots of organic matter and high pH are sustained for long periods of time in piles in which fresh material is added daily. The enhanced production of WMM could be explained by the onset of organic matter hydrolysis while still in the manure piles. Mink manure contains
Waste mink feed was the fourth of the substrates assessed. The specific methane production for this stream was 548 ± 33 mLCH4gVSadded corresponding to 91% of the theoretical bio methane production with a significant daily productivity of 65.0 mLCH4gVS-d. This productivity was the highest observed in the present study and is similar to that shown by Fabbri et al. [29] for the management of artichoke wastes while significantly lower to that reported by Zhou et al. [30] for the bio-methanation of bean curd wastes. The specific CH4 production of WF is higher compared to the bio methane potential of human food wastes while it is coming in par with substrates including fish feed wastes (527 mL), pasteurized blood (520 mL) and confectionary waste products (527 mL) [23]. Mink feed is rich in protein and fat and this is the reason why its production is much higher than the one expected to be recovered by human waste food of which cellulose and hemicellulose make up a significant portion. 3.4. pH and VFA development In all four assessed substrates, the pH had shown a small reduction up to 0.4 units during the first days of the experiments in response to the increasing concentrations and primary accumulation of the volatile fatty acids. This process however got reversed as the acids were consumed and converted into biogas by the methanogens. Accumulation of volatile fatty acids and reduction of the pH during the initial phases of the anaerobic digestion process is common, especially in batch systems, resulting from the lag and slow response of methanogens towards starting to consume the substrates provided to them by acidogens. 3.5. The effect of the loading rate The initial organic loading rate is an important parameter that must be considered during batch digestion. High loading rates may
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inhibit the process through either product or substrate accumulation; low loading rates may cause underestimation of the methane production potential of the substrates [31]. Three initial loading rates were assessed, namely 12.5, 25 and 50 gVS/L. While the highest OLR assessed appears as extreme, similar OLRs have been applied efficiently in the past for the management of food waste, animal manure, bone and meat meal and algal biomass [20,32,33]. Recently semi continuous systems have been operated efficiently up to 20 kgVSm3-d when treating easily acidified substrates including food waste [34]. In Fig. 2 the cumulative production of the different samples at the loading rates tested in this study is presented. Fig. 2A and B shows the bio methane potential for the FMM and WMM, - more specifically, the specific production for both substrates was reduced in response to the increase of the OLR. The reduction for the FMM reached 27 and 79 mL/gVSadded corresponding to reduced production of 7 and 21% respectively for the 25 and 50 gVS/L OLRs. With regard to the WMM the reduction registered as 60 and 149 mL/gVS equals to a lower production of 14 and 34% when compared to the bio methane potential achieved by the OLR of 12.5 gVS/L. Furthermore, the inhibitory effect for WMM was higher than the one for FMM. This could be related to the higher amount of soluble matter available in WMM. When this soluble matter comes in contact with acetogenic anaerobic microorganisms it is converted at a higher rate into VFA, possibly resulting in process overload. The inhibition observed in this work fits well to the values reported by Chae et al. [35] for the digestion of pig manure under different loading rates (5e40% V/V) and operating temperature regime (25 Ce35 C). The reduced production reported by these researchers were, 25% and 46% compared to the ultimate production (421 mLCH4/gVSadded) achieved under the loading rate of 10% V/V. Fig. 2C and D shows the effect that the different loading rates have on the anaerobic digestion of WF and the BMM. The increase of OLRs affected significantly the bio methane potential of the substrates. Reduction reached 129.7 mL and 151 mLCH4/gVSadded for the 25 gVS/L, corresponding to 24 and 25.5% for the WF and MBM respectively. When the OLR increased to 50 gVS/L a further reduction of the bio methane production was observed. In response to the increase of the OLR the methane production of the BMM was reduced by 491 mLCH4/gVSadded corresponding to a reduction of 83%. For the WF the bio methane potential got reduced by 384 mLCH4/gVSadded corresponding at a reduction of over 70%.
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Apart from the reduction in the cumulative methane production the daily methane productivity for all samples shows a negative response to the increase in OLRs. The greatest reduction was presented by BMM, where daily productivity got reduced from 64 mL, to 22 and 7 mL for the OLR of 12.5, 25 and 50 gVS/L respectively. The statistical difference for the OLRs was non-significant for FMM and WMM with p values > 0.05. In contrast, the different OLR produced data statistically significant for the WF and BMM with p values of <0.02 for both substrates. 3.6. VFA & pH pH levels and VFA concentrations are significant indicators of anaerobic digestion performance. pH and VFA are interrelated as an increase in VFA concentrations will inevitably affect pH. If the concentration of the acids exceeds the buffering capacity of the digester the process will be inhibited since pH will drop below the levels where methanogenesis occurs. In the present study a small pH reduction and accumulation of VFAs was registered in all experiments (results for the OLR of 12.5 kgVS/m3 are presented in section 3.1). Related to the OLR of 25 kgVS/m3 (Fig. 3), the batches treating the FMM and WMM showed a VFA accumulation of up to 2.7 and 3.8 g/L for the FMM and the WMM respectively. pH followed a similar trend with values descending up to 0.5 units. In all cases, more than 55% of acidic compounds was acetic acid. This accumulation however was reversible and the accumulated VFAs were converted into biogas followed by the recovery of the pH in the following days. When the OLR increased further to 50 kgVS/m3 the concentrations of VFAs reached 4.9 g/L and 5.1 g/L for the FMM and WMM respectively; pH was reduced by up to 0.8 units. Again this condition was reversible and the microorganisms were able to bio-convert the accumulated acids. Although during the digestion of FMM acetic acid was the most abundant acid, in the case of WMM the most abundant acid was propionic acid, making up approximately 60% of the TVFAs. When examining the WF, the process exhibited an accumulation of VFAs up to 4.1 g/L in the OLR of 25 kgVS/m3 which resulted in a pH reduction of about 0.7 units. When the OLR increased further, to 50 kgVS/m3, the pH dropped below 6 and the process was inhibited with very low daily methane productivity. The same trend was shown for the BMM, an initial accumulation of VFAs of up to 4.6 g/L was noticed. That was reversible and significant volumes of biogas were finally generated at the OLR of 25 kgVS/m3. At OLR of 50 kgVS/
Fig. 2. Cumulative methane production versus the total VFA development for the different organic loading rates. (A) FMM, (B) WMM, (C) WF, (D) BMM.
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m3 the concentrations of VFA accumulated reached 11.2 g/L thus triggering an irreversible process inhibition. In the mink-derived byproducts VFAs followed a similar trend as in the case of WMM with the acetic acid being the most abundant acid in the lower loading rate, while longer chain acids accumulated in the higher loading rates. Siegert and Banks [36] examined the effect that the addition of difference concentrations of VFA have on anaerobic digestion performance when cellulose and glucose are used as substrate. The VFA addition rate fluctuated between 1 and 20 g/L. According to these researchers a 65% reduction of the biogas production is caused by the addition of 4 g/L VFA when cellulose is used as substrate. In contrast to this, when glucose is digested, 14 g of VFAs are required to reproduce the same inhibitory effect. Furthermore, it is recognized that the reduced bio methane production driving process is related to the feedback inhibition of the hydrolysis stage. More specifically, intermediate products inhibit the enzyme or enzymes, responsible to activate specific metabolic pathways. Unfortunately, no data is provided related to the effect that the different concentrations of VFA had on the pH of the reactors, as the addition of 20 g/L VFAs could result in the reduction of the pH, within the reactors, below the levels required for methanogenesis. In the present study inhibition seems to be a synergistic effect based on the overloading of the process. Firstly, the generated VFAs overwhelmed the ability of the methanogens to consume them and convert them into biogas. This was followed by VFA accumulation and pH drop triggering process inhibition due to the inactivation of methanogens. An initial explanation why this behavior was seen only in the WF and BMM can be the higher acidification rates experienced in these two substrates due to their physical state. WF and BMM are either minced or milled offering in this way significant contact area for the microorganisms and enzymes to work on and convert them into organic acids. 3.7. The effect of the inoculum to substrate ratio Initial inoculum to substrate ratios are very important for the bio methane potential analysis of substrates. The selected rate should promote primarily a robust process, while at the same time it should minimize the interference of the endogenous biogas production from the inoculum. In order to identify the acceptance and compatibility of the substrates to anaerobic digestion systems, three I/S ratios were examined (equal to 2, 1 and 0.5 respectively). The cumulative biomethane production of the different samples is presented in Fig. 4 while Table 3 is providing a summary of the different productions and productivities. In all cases, the
reduction of the I/S ratio affected negatively the process with the higher reduction (33%) obtained from BMM (Fig. 4D), when the substrate was digested with an I/S ratio of 0.5. Likewise, a 31% reduction was observed for WF (4C), amounting to a reduction of 98 mL CH4gVSadded. In contrast to the high reduction of the methane production recorded for the two mink-derived byproducts, the reduction registered for the two types of manure tested was very small (less than 20%). This might well be the effect of process enhancement by microorganisms already established in the manure. While the ultimate production for I/S of 0.5 was similar to the values generated with I/S of 2, the rate by which production was achieved was very different. As shown in Fig. 4A and B, when manure was digested with I/S of 2, approximately 70% of the total biomethane was produced within the first week of the experiment. In contrast to this, when the I/S ratio was reduced to 1, it took approximately 12 days to achieve the same production. When the I/S ratio got reduced further more than 16 days were et al. [14] examined the required to achieve the same yield. Brule effect of the I/S ratio on the digestion of silage and green clippings by applying 3, 5 and 10-fold dilution of the inoculum, with an OLR of approximately 12 kgVSm3 and concluded that by providing trace elements into the process, the volume of inoculum was an insignificant process parameter affecting neither the cumulative yield nor the production rate. Without the addition of trace elements, a 15e40% reduction in methane yield and a long lag phase could be expected. Raposo et al. [37] examined the effect of 6 I/S ratios between 3 and 0.5 during anaerobic digestion of sunflower oil cake and concluded that by reducing the I/S ratio, the bio methane potential could be reduced by more than 60%. The same trend is presented by Dechrugsa et al. [38] when digesting grass and pig manure. These researchers further proposed that in case of dry substrates, I/S ratios over 4 are required in order to ensure the presence of significant populations of hydrolytic microorganisms to ensure a representative bio methane production. Indeed, in our study daily productivity was significantly lower for I/S of 0.5 while no significant accumulation of VFAs was registered. The lack of VFA accumulation indicates that at least methanogenesis was not the rate limiting stage of the process; methanogens were able to bio convert the acids offered to them by the acetogens. In all cases the statistical analysis of daily productivity for the 30 days revealed that the effect of the I/S ratios assessed was not significant with p values > 0.05. On the other hand, when considering only the first 10 days of the experiment, the statistical difference was significant with p values lower than 0.02 for all substrates and I/S ratios assessed.
Fig. 3. VFA fractionation for the four substrates and the three organic loading rates, results presenting the highest accumulated concentrations.
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7
Fig. 4. Cumulative methane production for the four substrates under the three inoculum to substrate ratios. (A) FMM, (B) WMM, (C) WF, (D) BMM.
Table 3 Summarizing table for the expected productions for the four substrates and the three organic loading rates. FMM Initial OLR kgVS/m3 Specific methane production (mLCH4/gVSadded) Daily productivity (mLCH4/gVSadded) Percentage of theoretical production (%) Volumetric CH4 production per ton product FM (m3)
WMM
WF
BMM
12.5
25
50
12.5
25
50
12.5
25
50
12.5
25
50
367.7 52.9 67.4 135.7
341.1 29.6 62.5 125.9
287.5 18.2 52.7 106.0
428.0 60.6 78.8 124.9
367.9 33.2 69.7 107.3
279.5 23.4 51.4 81.4
547.8 65.0 91.1 201.1
418.1 23.8 68.7 153.5
163.7 12.3 27.2 60.1
590.7 63.6 83.7 482.1
439.8 22.3 62.3 359.0
99.4 6.6 14.0 81.1
4. Conclusion In the present study the mesophilic anaerobic digestion process was successfully applied to 4 types of mink farm generated waste. The experimental results indicated that in contrast to pig and cattle manure, minks generate waste which may offer very high specific and volumetric methane productions. Thus, we deem that with proper management the economic viability of bio-methanation plants can ensured. The anaerobic digestion process lends itself toward mink-derived waste and byproducts. The process failed due to accumulation of intermediates only for the higher OLR assessed. On the other hand, even with diluted inoculum the process was successful and large volumes of methane was recovered. Further standardization is needed in the methods used to evaluate the performance of different substrates. A meaningful contribution in this direction from our study is the evaluation of the I/S ratio and its meaning, including the dilution of the substrate or of the inoculum as well as the increase or decrease of the initial loading rate of the system. References [1] F.a.t.M, 2012. Department of Agriculture, Report of the Fur Farming Review Group, Available On-line, https://www.agriculture.gov.ie/media/migration/ publications/2012 (assessed 10.10.15). [2] T. Zhang, H. Zhang, X. Wu, Q. Guo, Z. Liu, W. Qian, X. Gao, F. Yang, G. Li, Effects of dry dietary protein on digestibility, nitrogen-balance and growth performance of young male mink, Anim. Nutr. 1 (2) (2015).
€ lo € nen, P. Niemela €, Y. Xiao, L. Jalkanen, H. Korhonen, J. Ma €kela €, Formic [3] I. Po acidesodium benzoate preserved slaughterhouse offal and supplementary folic acid in mink diet, Anim. Feed Sci. Technol. 78 (1999) 39e56. [4] I. Hussain, G.W. Price, A.H. Farid, Inactivation of Aleutian mink disease virus through high temperature exposure in vitro and under field-based composting conditions, Vet. Microbiol. 173 (2014) 50e58. [5] R. Manchester, Fire and explosion hazards of meat & bone meal: storage, transport and processing, in: ICHEM E Symposium Series No. 149, 2003. [6] P.G. Kougias, T.A. Kotsopoulos, G.G. Martzopoulos, Effect of feedstock composition and organic loading rate during the mesophilic co-digestion of olive mill wastewater and swine manure, Renew. Energy 69 (2014) 202e207. [7] D.M. Wall, E. Allen, R. O'Shea, P. O'Kiely, J.D. Murphy, Investigating two-phase digestion of grass silage for demand-driven biogas applications: effect of particle size and rumen fluid addition, Renew. Energy 86 (2016) 1215e1223. [8] X. Fonoll, S. Astals, J. Dosta, J. Mata-Alvarez, Anaerobic co-digestion of sewage sludge and fruit wastes: evaluation of the transitory states when the cosubstrate is changed, Chem. Eng. J. 262 (2015) 1268e1274. [9] I.S. Zarkadas, A.S. Sofikiti, E.A. Voudrias, G.A. Pilidis, Thermophilic anaerobic digestion of pasteurised food wastes and dairy cattle manure in batch and large volume laboratory digesters: Focussing on mixing ratios, Renew. Energy 80 (2015) 432e440. [10] E. Elbeshbishy, G. Nakhla, H. Hafez, Biochemical methane potential (BMP) of food waste and primary sludge: Influence of inoculum pre-incubation and inoculum source, Bioresour. Technol. 110 (2012) 18e25. [11] M. Kawai, N. Nagao, N. Tajima, C. Niwa, T. Matsuyama, T. Toda, The effect of the labile organic fraction in food waste and the substrate/inoculum ratio on anaerobic digestion for a reliable methane yield, Bioresour. Technol. 157 (2014) 174e180. [12] M.R. Haider, Zeshan, S. Yousaf, R.N. Malik, C. Visvanathan, Effect of mixing ratio of food waste and rice husk co-digestion and substrate to inoculum ratio on biogas production, Bioresour. Technol. 190 (2015) 451e457. [13] A.G. Hashimoto, Effect of inoculum/substrate ratio on methane yield and production rate from straw, Biol. Wastes 28 (1989) 247e255. , R. Bolduan, S. Seidelt, P. Schlagermann, A. Bott, Modified batch [14] M. Brule anaerobic digestion assay for testing efficiencies of trace metal additives to
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Renewable Energy journal homepage: www.elsevier.com/locate/renene
Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process I. Zarkadas a, *, G. Dontis a, G. Pilidis b, D.A. Sarigiannis a a b
Aristotle University of Thessaloniki, Department of Chemical Engineering, Environmental Engineering Laboratory, 54124, Thessaloniki, Greece University of Ioannina, Department of Biological Application and Technology, University Campus, 45110, Ioannina, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2015 Received in revised form 11 February 2016 Accepted 15 March 2016 Available online xxx
Mink farming is a well-established economic activity with a significant environmental footprint. In this work mink farming derived organic waste was assessed, for the first time, as substrate to anaerobic digestion plants. The substrates assessed were; (a)fresh mink manures, (b)weathered mink manures, (c) waste feed and (d)mink derived meat and bone meal. Substrates with in inoculum to substrate ratio of 2 offered specific methane productions ranging between 368 and 591 mLCH4/gVSadded corresponding to 67.4 and 91.1% of their theoretical methane potential. In the second phase of the experiments three organic loading rates and three inoculum to substrate ratios were assessed. Substrate/inoculum ratios, in batch mode, lower than 1 seem to affect negatively the process, due to slow hydrolysis and acetogenesis of the macromolecules. In addition, initial organic loading rates of up to 50 gVS/L can be applied in batch systems when manure is utilized as substrate. In contrast to this, when mink derived byproducts are used the same loading rate will result into an irreversible process inhibition due to the accumulation of intermediate products. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic digestion Mink waste Bio methane production Farming waste Meat and bone meal Biogas production
1. Introduction European fur production is a dynamic and well established industry with long tradition in the production of quality products and significant exporting experience and capabilities. The European Union is the main exporter of pelts worldwide accounting for the 64% of the total production with the States of Denmark, Netherlands, Finland and Greece being the main producers [1]. Other significant fur producers are the United States of America and China, with China being the main exporter of fur derived commodities and clothing. The specie farmed for its fur is the American Mink (Neovison vison/Mustela vison) which is a carnivore, semiaquatic mammal [2], with the full grown males reaching the 45 cm in length, 20 cm in height and the 2 kg in weight. The diet of the farmed animals is composed of low cost protein and fat rich byproducts, including slaughterhouse and aquaculture wastes [3]. The wastes generated from mink breading facilities includes manure and waste feed. Both are collected under the animal cages in small piles of up to 50 cm in height. The waste management
* Corresponding author. E-mail addresses: [email protected] (I. Zarkadas), [email protected] (G. Dontis), [email protected] (G. Pilidis), [email protected] (D.A. Sarigiannis).
options for this waste stream are constrained by the high solids, organics and nitrogen content that are significantly hampering the abilities of aerobic biological processes to treat or valorize them. Even so, composting is widely employed, due to low cost [4]. Mink farmers in order to stimulate the application of the generated compost offer it free of charge. However, acceptance by local farmers and application rates to the fields are disappointing; bad odors one of the main reasons for this rejection. Manure stockpiling and composting on the other hand can promote nitrogen and phosphorus leachate and runoff formations leading to algae blooms and eutrophication of surface waters while, contamination of underground waters with fecal microorganisms is not uncommon. After pelting dead mink bodies are either sent for incineration or rendering where they are converted into bone and meat meal (BMM). The EC Animal By-product Regulation enforces the pretreatment of the dead minks through crushing, milling and cooking at 133 C and at least 3 bars of pressure for 20 min. The high protein meal after the process is used as soil amendment (fertilizer) or as alternative fuel. Unfortunately, this market is undeveloped and the ready product is stockpiled. Bone and meat meal on the other hand can become a hazardous commodity during both storage as well as transportation due to its tendency to self-heating and igniting without the requirement for an external source of heat or fire [5].
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Abbreviations TOC TS AD HRT FMM WMM WF BMM
Total Organic Carbon Total Solids Anaerobic Digestion Process Hydraulic Retention Time Fresh Mink Manure Weathered Mink Manure Waste Mink Feed Mink Derived Bone and Meat Meal
A waste management option which can be employed for the valorization of fur farming wastes is anaerobic digestion (AD). AD is a biomass bio-conversion process disengaged from weather conditions which offers the advantages of self-sustainability, income generation and waste valorization with limited material requirements. Anaerobic digestion of manures and plant waste material offers the opportunity for recovery of both biogas (a methane rich gas) and hygienized and nearly odorless stabilized digestate which can be applied onto agricultural land as an organic fertilizer. Anaerobic digestion has been assessed and applied to a plethora of different substrates/wastes/low value or negative value by products including animal manures [6], industrial wastes and wastewaters, municipal solid wastes, energy crops [7] and mixtures of different substrates in co-digestion schemes [8,9]. The substrates evaluated in this work were fresh and weathered mink manures (FMM, WMM respectively), waste mink feed (WF) and bone and meat meal (BMM) that is being generated by the mink carcasses after pelting. The scientific community has shown very limited interest for the management of the mink farming waste and byproducts. This can be due either to the absence of these operations in most of the developed world or due to the negative publicity that these operations have as an effect of the number of animals sacrificed so that their furs become high value human clothing and accessories. This is the first time that the mink farming waste is assessed as candidate substrate to anaerobic digestion process. 2. Material and methods 2.1. Substrates and inoculum The substrates selected included all important waste streams generated during the life cycle of minks. Unfortunately seasonality is a significant problem as most of the wastes are generated between May and November. During winter and early spring only low volumes of manure and waste feed are produced by the males and the breading females. The substrates were assessed in batch vials under mesophilic conditions mainly due to the known problems related to the inhibition of the process by elevated concentrations of unionized ammonia. This inhibitory process is driven by the bio-conversion, of protein into ammonia and it is assisted by the high temperature and pH experienced in thermophilic systems. Inoculum was collected from two 6 L laboratory scale CST reactors treating mixtures of cattle manure and food waste and operating under an OLR of 3.1 kgVSm3R-d and a HRT of 28 days. Prior to the preparation of the batches the inoculum was degassed for 1 week. Mink manure was collected from a medium size farm housing approximately 20.000 minks and located in the area of Kastoria. Fresh mink manure was considered the manure located at the upper part of the pile that kept its as excreted shape; weathered manure was considered the one located inside the piles, which had
lost its shape and its physical appearance had been altered into a thick paste. The smell of both substrates were strong and unpleasant. Waste feed was collected from the same farm and composed by the uneaten feed provided on top of each mink cage. BMM was collected from a rendering operation which is responsible for the disposal, during the pelting season, of the mink bodies and operating according to the EC Animal Byproduct Regulation. In this facility waste fat is recovered and sent for biodiesel production, while BMM is sold as fertilizer or as renewable fuel. 2.2. Batch preparation During the preparation of the batches, the vials were filled with the inoculum, the substrates and distilled water was used as filler to the active volume of 120 mL. Finally the oxygen removed through nitrogen bubbling and the batches were placed within a tempera ture controlled cabinet at 36 ± 1 C for 30 days. The same steps were used for the preparation of the batches utilized for the assessment of the different I/S ratios except that instead of water, 0.02 M potassium phosphate buffer (pH 7.2) was used for inoculum dilution. The volumetric gas production assessed daily, pH and VFA concentrations analyzed every 3e5 days and all experiments took place in triplicate. In this work three inoculum to substrate ratios 2,1, 0.5 (initial OLR of 12.5 kgVSm3) as well as three organic loading rates have been assessed. The data related to the batch preparation are presented in Table 1. To date there is a lack of standard methodologies for the application of bio methane potential test [10]. The assessment of the effect of the inoculum to substrate ratio in the digestion of different substrates is done through stepwise increase of the organic loading [11,12]. Although this is technically correct, in many cases, especially when high solid substrates are assessed, it offers data related to the tolerance of the process in extreme loading rates not applicable in real life digestion systems. In this work the idea of keeping constant the OLR and diluting, with a pH buffer, the inoculum added into the systems has also been assessed. Such an approach has been used in the past by Hashimoto [13], and et al. [14] when examining the biodegradability of straw, Brule silage and green clippings. The dilution of the inoculum is affecting a number of operational parameters, including the number of the available cells and the concentration of trace elements [14]. At the same time the presence of an inhibitor might be revealed through this stepwise process. Successful digestion demonstrates the acceptability of the substrate by the process. 2.3. Analytical methods 2.3.1. Volatile fatty acids, TOC, TKN, pH, soluble COD & biogas quality, ammonia For VFA analysis samples were centrifuged to 9000 RPM for 10 min and the supernatant were analyzed with a Shimadzu
Table 1 Composition of the experimental series. Experimental series
OLR 12.5 kgVS/m3
OLR 25 kgVS/m3
OLR 50 kgVS/m3
FMM(g FM) WMM(g FM) WF (g FM) BMM(g FM)
4.08 5.16 4.10 1.84 I/S 2 3.01 4.08 5.16 4.10 1.84
8.17 10.33 8.21 3.69 I/S 1 1.50 4.08 5.16 4.10 1.84
16.34 20.66 16.42 7.39 I/S 0.5 0.75 4.08 5.16 4.10 1.84
Inoculum (gVS) FMM(g FM) WMM(g FM) WF(g FM) BMM(g FM)
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I. Zarkadas et al. / Renewable Energy xxx (2016) 1e8
GC17A Gas Chromatographer with a flame ionization detector (GC-FID). For the separation of VFA's a Nukol 15 m 0.53 mm column was used. The volume of the generated gas was measured according to the liquid displacement principle as described by Zarkadas and Pilidis [15] with the application of inverted cylinders. Sodium hydroxide 2N was used for carbon dioxide scrubbing with the concentration of methane to be validated with a Shimadzu GC 2014 coupled to a thermal conductivity detector. A CARBOXEN 1000 60/80 Column of 15 ft 1/8in was used. TOC analysis conducted with a Shimadzu TOC-VCPH carbon analyzer coupled to a solid state combustion unit- SSM-5000A, total organic carbon concentrations were obtained by deducing the inorganic carbon from the total carbon readings. Total Solids and Volatile solids analyzed as described by Standard Methods, (APHA 1989). Total Kjeldahl Nitrogen was analyzed with a HACH Digesdahl Digestion Apparatus and the method 8075. pH analysis was conducted with a Hanna pH 211 microprocessor pH meter. For soluble COD analysis the samples were diluted with distilled water addition in a ratio of 1/1, followed by manual mixing and the samples filtered through 0.45 mm pore size filters with the utilization of vacuum vials and a hand pump. The analysis was conducted with the application of HACH COD cuvettes and a DR2000 direct reading photometer. The ammonia concentration of the manure was analyzed using a Hanna HI 84185 coupled to an ammonia selective electrode. The theoretical methane production calculated according to[16] and the application of the following conversion formula: CH4m3/kgVS ¼ (0.496X) þ (1.014Y) þ (0.415Z)
Where: X ¼ % Protein fraction of VS Y ¼ % Fat fraction of VS Z ¼ % Carbohydrates fraction of VS
2.3.2. Statistical analysis The significance of differences in methane yield and content determined by applying single factor Analysis of Variance (ANOVA) in MATLAB R2012b. Statistical significance was established at a PValue of less than 0.05. 3. Results and discussion In this study four types of mink farming waste, were assessed as substrates to mesophilic anaerobic digestion. Their characteristics are given in Table 2. The substrates are in the solid state with TS higher than 33%. The solids are composed mainly of organic matter with VS levels higher than 83%. They have significant nitrogen content with TKN concentrations ranging between 14 and 93 g/Kg, while the pH of the manure is alkaline. This is in contrast to that of
3
the WF, which is acidic and that of BMM, which is neutral. Finally the theoretical methane production of the substrates range between 545 and 705 mLCH4/gVSadded. The experiments were divided into two stages. In the first part the bio methane potential of the different substrates were investigated in an inoculum to substrate ratio of 2 and an OLR of 12.5 kgVS/m3, as these operational parameters have been shown by other researches [17,18] as appropriate for: a) providing the required number of microorganisms that will initiate a robust process b) minimizing the likelihood of overloading the process by intermediate products accumulation, including the volatile fatty acids, and c) reducing latency and occurrence of an initial lag phase. In the second part of the experiment, two additional inoculum to substrate ratios were examined with another two organic loading rates. Low initial inoculum loadings were examined in order to identify and assess the compatibility and acceptance of the substrates by the anaerobic digestion microorganisms. 3.1. Biomethane potential Fig. 1 shows the cumulative and daily methane production of the different substrates. Methane production started rapidly without the presence of a lag phase. The highest production was observed by BMM with 591 ± 38 mL/gVSadded, a value corresponding to the 83.7% of the theoretical methane production for this substrate. Bone and meat meal sourced from different facilities have been assessed in the past in relation to their bio methane potentials and the possibility of these products to be used as substrates to anaerobic digestion plants. Most frequently BMM available on the market is produced from waste generated during the slaughtering of farmed animals. This waste together with blood, feather and bone meals are the byproducts of slaughtering and dressing animals ready for human consumption. In all cases the soft edible tissues are removed and sold for human consumption, while the BMM is produced from the offal, condemned meat and the bones. Mink BMM includes also soft tissue as it is not possible for the mink meat to be utilized further and it must be disposed or valorized through processes alternative to human or pet animal food/feed manufacturing. The bio methane potential of the BMM assessed in the present study was significantly higher compared to the values found in the relevant literature [19,20]. There specific methane production fluctuates between 225 and 390 mLgVSadded. An explanation to the significantly higher production of the mink derived BMM might be the difference in the fat and protein content of the products assessed. In the marketable bone and meat meal, fat content fluctuates between 12 and 14% in agreement with the literature value of 11.9% reported by Hejnfelt and Angelidaki [19].
Table 2 Characterization of the substrates utilized (N/A-not analyzed).
Total solids (%) Volatile solids (%TS) Total Kjeldahl nitrogen g/kg NH3-N (g/kg) Total organic carbon (%TS) Total fat (%TS) pH Soluble COD (g/kg) Theoretical CH4 production mLCH4gVSadded
Inoc.
FMM
WMM
WF
BMM
5.12 80.7 1.69 N/A 30.4 0.26 7.81 N/A e
41.8 88.3 17.9 5.74 43.8 7.52 9.12 14.5 545.5
33.9 86.1 14.4 4.96 41.6 5.19 8.53 25.9 543.0
39.7 92.5 31.2 N/A 44.6 16.9 5.90 N/A 601.0
97.4 83.8 93.1 N/A 47.2 21.4 7.12 N/A 705.9
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Fig. 1. (A) Daily specific methane production, (B) cumulative methane production versus total VFA development.
straw, which is provided to the animal as bedding material. Cellulose and especially its crystalline form is one of the most difficult natural macromolecules for degradation under anaerobic conditions [25]. During sampling and preparation of the experiments in the FMM long straws were visible, unlike what happened in the case of WMM where only short straw pieces were visible; the latter were much softer and easily crashed by hand. Furthermore, mink manure is rich in ammonia, a chemical known to be used during pretreatment of straw or cellulose rich substrates prior to anaerobic digestion or bioethanol production per se [26,27]. Ammonia application and pretreatment promotes hydrolysis and release of simple cellulose sugars deprived of the negative effects of the lignin which is not converted anaerobically. Mink manure is formed by the animals in piles, which are removed twice per year. The in-pile retention time of the manure in the presence of ammonia can promote straw hydrolysis, and in turn improve conversion efficiency and bio-methane production. Another plausible mechanism leading to enhanced hydrolysis could be micro aeration [28]. The anoxic conditions formed promote microorganisms that increase the solution of the substrate. A third possible mechanism could be the weathering of an inhibitor present in the FMM but not in the WMM is also possible, however no such inhibitor was identified in the present study. The daily methane production was also higher for the WMM compared to the FMM with the daily productivity, during the exponential phase, averaging at 52.9 and 60.6 mLCH4gVSadded for the FMM and the WMM respectively. Thus WMM resulted in a 13% higher bio-methane production than FMM. 3.3. Waste mink feed
In contrast to this the fat content of BMM in our study was higher than 20%. Considering that the bio methane potential of fat is roughly twice that of both carbohydrates and proteins, the increased values achieved could be the effect of the high fat content in the substrate. Furthermore the bone and meat meal used in other literature studies had at least 10% more protein, rendering the formation of ammonia and the resulting process inhibition a formidable problem. Daily methane production during the exponential phase for the BMM was found to mount to 63.6 mL/gVSadded presenting two production peaks on days 4 and 8, probably due to the different bio-conversion rates of the substrate components. 3.2. Fresh and weathered mink manure The bio methane potential of the mink manure was 368 and 428 mLCH4gVSadded for the fresh and weathered manures respectively. Mink as a monogastric carnivore requires feed rich in protein and fat while its digestion track efficiency fluctuates between 68.9 and 74.3% with the fat being readily digested with up to 90% [2]. The bio methane potential of mink manure is significantly higher when compared to common anaerobic digestion substrates including the manure generated by pigs, cattle or poultry; the latter rarely exceed the specific methane production of 300 mLCH4gVSadded [21,22]. Thus, the bio methane potential of mink manure is at par with high quality food waste [23] and slaughterhouse waste [24]. The bio methane potential of WMM was 14% higher than that of FMM. This is counterintuitive, since in general weathering affects negatively the quality of the substrates as part of it is lost as carbon dioxide to the atmosphere. In the case of mink manure, materials with lots of organic matter and high pH are sustained for long periods of time in piles in which fresh material is added daily. The enhanced production of WMM could be explained by the onset of organic matter hydrolysis while still in the manure piles. Mink manure contains
Waste mink feed was the fourth of the substrates assessed. The specific methane production for this stream was 548 ± 33 mLCH4gVSadded corresponding to 91% of the theoretical bio methane production with a significant daily productivity of 65.0 mLCH4gVS-d. This productivity was the highest observed in the present study and is similar to that shown by Fabbri et al. [29] for the management of artichoke wastes while significantly lower to that reported by Zhou et al. [30] for the bio-methanation of bean curd wastes. The specific CH4 production of WF is higher compared to the bio methane potential of human food wastes while it is coming in par with substrates including fish feed wastes (527 mL), pasteurized blood (520 mL) and confectionary waste products (527 mL) [23]. Mink feed is rich in protein and fat and this is the reason why its production is much higher than the one expected to be recovered by human waste food of which cellulose and hemicellulose make up a significant portion. 3.4. pH and VFA development In all four assessed substrates, the pH had shown a small reduction up to 0.4 units during the first days of the experiments in response to the increasing concentrations and primary accumulation of the volatile fatty acids. This process however got reversed as the acids were consumed and converted into biogas by the methanogens. Accumulation of volatile fatty acids and reduction of the pH during the initial phases of the anaerobic digestion process is common, especially in batch systems, resulting from the lag and slow response of methanogens towards starting to consume the substrates provided to them by acidogens. 3.5. The effect of the loading rate The initial organic loading rate is an important parameter that must be considered during batch digestion. High loading rates may
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inhibit the process through either product or substrate accumulation; low loading rates may cause underestimation of the methane production potential of the substrates [31]. Three initial loading rates were assessed, namely 12.5, 25 and 50 gVS/L. While the highest OLR assessed appears as extreme, similar OLRs have been applied efficiently in the past for the management of food waste, animal manure, bone and meat meal and algal biomass [20,32,33]. Recently semi continuous systems have been operated efficiently up to 20 kgVSm3-d when treating easily acidified substrates including food waste [34]. In Fig. 2 the cumulative production of the different samples at the loading rates tested in this study is presented. Fig. 2A and B shows the bio methane potential for the FMM and WMM, - more specifically, the specific production for both substrates was reduced in response to the increase of the OLR. The reduction for the FMM reached 27 and 79 mL/gVSadded corresponding to reduced production of 7 and 21% respectively for the 25 and 50 gVS/L OLRs. With regard to the WMM the reduction registered as 60 and 149 mL/gVS equals to a lower production of 14 and 34% when compared to the bio methane potential achieved by the OLR of 12.5 gVS/L. Furthermore, the inhibitory effect for WMM was higher than the one for FMM. This could be related to the higher amount of soluble matter available in WMM. When this soluble matter comes in contact with acetogenic anaerobic microorganisms it is converted at a higher rate into VFA, possibly resulting in process overload. The inhibition observed in this work fits well to the values reported by Chae et al. [35] for the digestion of pig manure under different loading rates (5e40% V/V) and operating temperature regime (25 Ce35 C). The reduced production reported by these researchers were, 25% and 46% compared to the ultimate production (421 mLCH4/gVSadded) achieved under the loading rate of 10% V/V. Fig. 2C and D shows the effect that the different loading rates have on the anaerobic digestion of WF and the BMM. The increase of OLRs affected significantly the bio methane potential of the substrates. Reduction reached 129.7 mL and 151 mLCH4/gVSadded for the 25 gVS/L, corresponding to 24 and 25.5% for the WF and MBM respectively. When the OLR increased to 50 gVS/L a further reduction of the bio methane production was observed. In response to the increase of the OLR the methane production of the BMM was reduced by 491 mLCH4/gVSadded corresponding to a reduction of 83%. For the WF the bio methane potential got reduced by 384 mLCH4/gVSadded corresponding at a reduction of over 70%.
5
Apart from the reduction in the cumulative methane production the daily methane productivity for all samples shows a negative response to the increase in OLRs. The greatest reduction was presented by BMM, where daily productivity got reduced from 64 mL, to 22 and 7 mL for the OLR of 12.5, 25 and 50 gVS/L respectively. The statistical difference for the OLRs was non-significant for FMM and WMM with p values > 0.05. In contrast, the different OLR produced data statistically significant for the WF and BMM with p values of <0.02 for both substrates. 3.6. VFA & pH pH levels and VFA concentrations are significant indicators of anaerobic digestion performance. pH and VFA are interrelated as an increase in VFA concentrations will inevitably affect pH. If the concentration of the acids exceeds the buffering capacity of the digester the process will be inhibited since pH will drop below the levels where methanogenesis occurs. In the present study a small pH reduction and accumulation of VFAs was registered in all experiments (results for the OLR of 12.5 kgVS/m3 are presented in section 3.1). Related to the OLR of 25 kgVS/m3 (Fig. 3), the batches treating the FMM and WMM showed a VFA accumulation of up to 2.7 and 3.8 g/L for the FMM and the WMM respectively. pH followed a similar trend with values descending up to 0.5 units. In all cases, more than 55% of acidic compounds was acetic acid. This accumulation however was reversible and the accumulated VFAs were converted into biogas followed by the recovery of the pH in the following days. When the OLR increased further to 50 kgVS/m3 the concentrations of VFAs reached 4.9 g/L and 5.1 g/L for the FMM and WMM respectively; pH was reduced by up to 0.8 units. Again this condition was reversible and the microorganisms were able to bio-convert the accumulated acids. Although during the digestion of FMM acetic acid was the most abundant acid, in the case of WMM the most abundant acid was propionic acid, making up approximately 60% of the TVFAs. When examining the WF, the process exhibited an accumulation of VFAs up to 4.1 g/L in the OLR of 25 kgVS/m3 which resulted in a pH reduction of about 0.7 units. When the OLR increased further, to 50 kgVS/m3, the pH dropped below 6 and the process was inhibited with very low daily methane productivity. The same trend was shown for the BMM, an initial accumulation of VFAs of up to 4.6 g/L was noticed. That was reversible and significant volumes of biogas were finally generated at the OLR of 25 kgVS/m3. At OLR of 50 kgVS/
Fig. 2. Cumulative methane production versus the total VFA development for the different organic loading rates. (A) FMM, (B) WMM, (C) WF, (D) BMM.
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I. Zarkadas et al. / Renewable Energy xxx (2016) 1e8
m3 the concentrations of VFA accumulated reached 11.2 g/L thus triggering an irreversible process inhibition. In the mink-derived byproducts VFAs followed a similar trend as in the case of WMM with the acetic acid being the most abundant acid in the lower loading rate, while longer chain acids accumulated in the higher loading rates. Siegert and Banks [36] examined the effect that the addition of difference concentrations of VFA have on anaerobic digestion performance when cellulose and glucose are used as substrate. The VFA addition rate fluctuated between 1 and 20 g/L. According to these researchers a 65% reduction of the biogas production is caused by the addition of 4 g/L VFA when cellulose is used as substrate. In contrast to this, when glucose is digested, 14 g of VFAs are required to reproduce the same inhibitory effect. Furthermore, it is recognized that the reduced bio methane production driving process is related to the feedback inhibition of the hydrolysis stage. More specifically, intermediate products inhibit the enzyme or enzymes, responsible to activate specific metabolic pathways. Unfortunately, no data is provided related to the effect that the different concentrations of VFA had on the pH of the reactors, as the addition of 20 g/L VFAs could result in the reduction of the pH, within the reactors, below the levels required for methanogenesis. In the present study inhibition seems to be a synergistic effect based on the overloading of the process. Firstly, the generated VFAs overwhelmed the ability of the methanogens to consume them and convert them into biogas. This was followed by VFA accumulation and pH drop triggering process inhibition due to the inactivation of methanogens. An initial explanation why this behavior was seen only in the WF and BMM can be the higher acidification rates experienced in these two substrates due to their physical state. WF and BMM are either minced or milled offering in this way significant contact area for the microorganisms and enzymes to work on and convert them into organic acids. 3.7. The effect of the inoculum to substrate ratio Initial inoculum to substrate ratios are very important for the bio methane potential analysis of substrates. The selected rate should promote primarily a robust process, while at the same time it should minimize the interference of the endogenous biogas production from the inoculum. In order to identify the acceptance and compatibility of the substrates to anaerobic digestion systems, three I/S ratios were examined (equal to 2, 1 and 0.5 respectively). The cumulative biomethane production of the different samples is presented in Fig. 4 while Table 3 is providing a summary of the different productions and productivities. In all cases, the
reduction of the I/S ratio affected negatively the process with the higher reduction (33%) obtained from BMM (Fig. 4D), when the substrate was digested with an I/S ratio of 0.5. Likewise, a 31% reduction was observed for WF (4C), amounting to a reduction of 98 mL CH4gVSadded. In contrast to the high reduction of the methane production recorded for the two mink-derived byproducts, the reduction registered for the two types of manure tested was very small (less than 20%). This might well be the effect of process enhancement by microorganisms already established in the manure. While the ultimate production for I/S of 0.5 was similar to the values generated with I/S of 2, the rate by which production was achieved was very different. As shown in Fig. 4A and B, when manure was digested with I/S of 2, approximately 70% of the total biomethane was produced within the first week of the experiment. In contrast to this, when the I/S ratio was reduced to 1, it took approximately 12 days to achieve the same production. When the I/S ratio got reduced further more than 16 days were et al. [14] examined the required to achieve the same yield. Brule effect of the I/S ratio on the digestion of silage and green clippings by applying 3, 5 and 10-fold dilution of the inoculum, with an OLR of approximately 12 kgVSm3 and concluded that by providing trace elements into the process, the volume of inoculum was an insignificant process parameter affecting neither the cumulative yield nor the production rate. Without the addition of trace elements, a 15e40% reduction in methane yield and a long lag phase could be expected. Raposo et al. [37] examined the effect of 6 I/S ratios between 3 and 0.5 during anaerobic digestion of sunflower oil cake and concluded that by reducing the I/S ratio, the bio methane potential could be reduced by more than 60%. The same trend is presented by Dechrugsa et al. [38] when digesting grass and pig manure. These researchers further proposed that in case of dry substrates, I/S ratios over 4 are required in order to ensure the presence of significant populations of hydrolytic microorganisms to ensure a representative bio methane production. Indeed, in our study daily productivity was significantly lower for I/S of 0.5 while no significant accumulation of VFAs was registered. The lack of VFA accumulation indicates that at least methanogenesis was not the rate limiting stage of the process; methanogens were able to bio convert the acids offered to them by the acetogens. In all cases the statistical analysis of daily productivity for the 30 days revealed that the effect of the I/S ratios assessed was not significant with p values > 0.05. On the other hand, when considering only the first 10 days of the experiment, the statistical difference was significant with p values lower than 0.02 for all substrates and I/S ratios assessed.
Fig. 3. VFA fractionation for the four substrates and the three organic loading rates, results presenting the highest accumulated concentrations.
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Fig. 4. Cumulative methane production for the four substrates under the three inoculum to substrate ratios. (A) FMM, (B) WMM, (C) WF, (D) BMM.
Table 3 Summarizing table for the expected productions for the four substrates and the three organic loading rates. FMM Initial OLR kgVS/m3 Specific methane production (mLCH4/gVSadded) Daily productivity (mLCH4/gVSadded) Percentage of theoretical production (%) Volumetric CH4 production per ton product FM (m3)
WMM
WF
BMM
12.5
25
50
12.5
25
50
12.5
25
50
12.5
25
50
367.7 52.9 67.4 135.7
341.1 29.6 62.5 125.9
287.5 18.2 52.7 106.0
428.0 60.6 78.8 124.9
367.9 33.2 69.7 107.3
279.5 23.4 51.4 81.4
547.8 65.0 91.1 201.1
418.1 23.8 68.7 153.5
163.7 12.3 27.2 60.1
590.7 63.6 83.7 482.1
439.8 22.3 62.3 359.0
99.4 6.6 14.0 81.1
4. Conclusion In the present study the mesophilic anaerobic digestion process was successfully applied to 4 types of mink farm generated waste. The experimental results indicated that in contrast to pig and cattle manure, minks generate waste which may offer very high specific and volumetric methane productions. Thus, we deem that with proper management the economic viability of bio-methanation plants can ensured. The anaerobic digestion process lends itself toward mink-derived waste and byproducts. The process failed due to accumulation of intermediates only for the higher OLR assessed. On the other hand, even with diluted inoculum the process was successful and large volumes of methane was recovered. Further standardization is needed in the methods used to evaluate the performance of different substrates. A meaningful contribution in this direction from our study is the evaluation of the I/S ratio and its meaning, including the dilution of the substrate or of the inoculum as well as the increase or decrease of the initial loading rate of the system. References [1] F.a.t.M, 2012. Department of Agriculture, Report of the Fur Farming Review Group, Available On-line, https://www.agriculture.gov.ie/media/migration/ publications/2012 (assessed 10.10.15). [2] T. Zhang, H. Zhang, X. Wu, Q. Guo, Z. Liu, W. Qian, X. Gao, F. Yang, G. Li, Effects of dry dietary protein on digestibility, nitrogen-balance and growth performance of young male mink, Anim. Nutr. 1 (2) (2015).
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Please cite this article in press as: I. Zarkadas, et al., Exploring the potential of fur farming wastes and byproducts as substrates to anaerobic digestion process, Renewable Energy (2016), http://dx.doi.org/10.1016/j.renene.2016.03.056