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Biohydrogen production from autoclaved fruit and vegetable wastes by dry fermentation under thermophilic condition
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Biohydrogen production from autoclaved fruit and vegetable wastes by dry fermentation under thermophilic condition Haris Nalakath Abubackar a,b, Tugba Keskin a, Okyanus Yazgin a, Bensu Gunay a, Kubra Arslan a, Nuri Azbar a,* a
Bioengineering Department, Faculty of Engineering, Ege University, 35100, Izmir, Turkey Chemical Engineering Laboratory, Faculty of Sciences and Centre for Advanced Scientific Research (CICA), ~ a, Ru´a da Fraga 10, E e 15008 A Corun ~ a, Spain University of La Corun
b
article info
abstract
Article history:
Dark fermentative hydrogen production from organic waste is an attractive technique that
Received 15 October 2018
simultaneously treats waste along with generation of renewable fuel. In this study, a
Received in revised form
relative new technology named dark dry fermentation was tested in a 55-L reactor to treat
29 November 2018
fruit and vegetable waste (FVW) along with simultaneous generation of biohydrogen. To
Accepted 8 December 2018
understand the effect of autoclaving as a pretreatment method on FVW for subsequent
Available online 8 January 2019
biohydrogen production, two independent experiments were performed; one with autoclaved waste (experiment I) and another by using non autoclaved waste (experiment II).
Keywords:
From the analyses, it was found that maximum hydrogen % obtained for experiment I was
Biohydrogen
41% (v/v%) whereas, for experiment II was 21%. In terms of total hydrogen produced,
Autoclaving
around 30% higher production was observed with experiment I compared to experiment II.
Fruit and vegetable waste
The hydrogen yields for experiment I and experiment II were respectively, 27.19 and 20.81
Bioenergy
NmL H2/gVS (VS ¼ volatile solid added), and the metabolites (VFAs) preferentially produced were acetic acid and iso-butyric acid. © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction The worldwide generation of organic fraction of municipal solid waste (OFMSW) is estimated to be around 1012 million metric tons per year by 2025 [1]. These biodegradable wastes are generally disposed by landfilling or by open dumping. One way to treat them properly together with simultaneous generation of energy is by applying anaerobic digestion [2]. This not only mitigates the climate change caused from the greenhouse gas emissions but also helps to achieve
sustainability in terms of energy. Several countries set targets to reduce the emission of environmentally harmful gases. In this respect, European Environment Agency target to achieve a reduction of 40% greenhouse gas emissions cuts by 2030. According to the revised European Directive 2009/28/EC, by 2030, 27% renewable energy consumption should be achieved. Overall, to meet these requirements, a huge leap in research has been noticed for the past decade to find an alternative renewable energy source [3,4]. Hydrogen is considered as a promising energy source in terms of its high energy content (143 MJ/kg) compared to any
* Corresponding author. E-mail address: [email protected] (N. Azbar). https://doi.org/10.1016/j.ijhydene.2018.12.068 0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
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existing fuel, and its combustion generates water as the only by-product. To date, hydrogen was produced mainly from fossil fuels with 48% from natural gas, 30% from heavy oils and naphtha, 18% from coal, and 4% from electrolysis [5]. Recently hydrogen gas generation from renewable sources such as biomass, which is organic materials derived from plants and animals, is gaining much attention [6]. Biomass can be thermo-chemically or biologically treated to generate hydrogen where former mainly involves pyrolysis or gasification and catalytic steam reforming. Biological technologies include direct and indirect biophotolysis using algae and cyanobacteria, photo-fermentation by photosynthetic bacteria and dark fermentation by fermentative bacteria, and microbial electrolysis cell [7,8]. Dark fermentative bacteria use carbohydrate rich substrates and oxidise them releasing electrons, which under anaerobic or anoxic, dark conditions accepted by protons and thereby generating molecular H2. This is accompanied by the formation of various organic acids [9]. As a model substrate, glucose, upon fermentation produces mainly acetic acid and butyric acid along with a theoretical yield of 4 and 2 mol of H2, respectively [10]. It is considered as a promising technology in terms of its practicality to utilize different types of organic wastes as feedstocks and thus can also able to improve the economics of the process. Potential biomass waste for biohydrogen includes agricultural residue, industrial residue, livestock waste, OFMSW [11,12]. These lignocellulosic materials are recalcitrant for biodegradation unless suitable pretreatment methods have been applied. Several pretreatment methods have been developed and their effects on biogas and biohydrogen production were evaluated during the past decades [13,14]. Autoclaving is considered to be an ideal method as it offers several advantages over other physicochemical methods such as requirement of no chemicals and could able to preserve the nutrition present in the waste [15]. An ideal feedstock for biohydrogen should be rich in carbohydrate and deficient in nitrogen [16]. Fruit and vegetable wastes (FVW), which accounts for 45% of the human food lost, is an ideal candidate for fermentation in terms of its moisture content, biodegradability, low total solid and high volatile solid content [17]. Several studies have been undergone using fruit and vegetables by anaerobic digestion for biogas production [2,18] and by dark fermentation for biohydrogen production [19e21]. However, due the complexity of the structure, microorganisms are less access to the biodegradable substrate causing a reduction in the overall productivity from the process. Thus, waste has to be subjected to pretreatment for efficient cellulose digestion [22]. The process temperature is one of the main parameters that influence the biohydrogen production by dark fermentation. Operating under thermophilic condition (42e75 C) has several advantages over mesophilic condition including the inhibition of the hydrogen consuming microorganisms and possibility to treat directly the hot industrial effluents [10,23]. Anaerobic digestion can be classified into: wet fermentation where the total solid content is less than 10% and dry fermentation with total solid content greater than 10%. Dry fermentation is a promising technology due to its several advantages like requirement of smaller reactor volume, easiness in handling digestate and lesser water wastage [24e26].
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Although many studies have performed for the biogas production through dry fermentation still not much studies on biohydrogen production using this technology. In this study, dry fermentation studies were performed under thermophilic condition to understand the effect of pretreatment (autoclaving) of FVW on biohydrogen production. To the best of authors’ knowledge this study is a novel in terms of combining various factors such as a pretreatment applied, substrate utilized and fermentation temperature in dry fermentation system.
Experimental section Feedstock and inoculum The FVW was obtained from the Izmir municipality bazar (Turkey) and were either chopped into very small pieces using a grinder or cut into small pieces of size < 5 cm. The composition of FVW collected, expressed in mass percentage (wt%), were: radish (5.71), pepper (5.06), pomegranate (7.02), pear (3.33), apple (4.66), pumpkin (2.06), mandarin (1.68), tomato (7.45), onion (6.20), potato (5.48), peach (12.90), lemon (2.28), eggplant (11.34), carrot (8.68), orange (2.59), cucumber (6.70), cabbage (4.19), grape (2.68). The same FVW composition was used for all the experimental studies conducted in dry fermentor (DF) and reported in this manuscript. The inoculum was collected from a local biogas plant that uses dairy products and local animal wastes as feed (Izmir, Turkey) and underwent a pre-treatment at 105 C for 10 min in order to gather hydrogen producers. The characteristics of the pretreated inoculum used in these studies were: pH 9.45, soluble COD of 9000 mg/L, total sugar of 900 mg/L, ammonium concentration of 3610 mg/L, TS (total solid) and VS of 7.9 and 4.8%, respectively.
Dry fermentation system The experiments were performed in a fully automated stirred tank (ST) single walled reactor of a total volume of 13 L (INFORS HT Labfors 5 reactor, Switzerland) which contained hydrogen producing microbes and a 55-L custom-made reactor (DF) where the FVW and hydrogen producers were mixed and filled for treatment (Fig. 1). Temperature of the ST reactor was controlled via an inbuilt platinum resistor temperature sensor (pt100) that was inserted in a blink pocket of the reactor and by using cold finger to circulate the cooling water. The dry fermentor was fabricated from an autoclave unit and an aluminum basket with holes around the sides and bottoms placed inside the unit for introducing the FVW and holding it, and allowing the leachate to flow freely to the bottom of the dry fermentor unit to the leachate collection tube. The inside temperature of the dry fermentor unit was controlled by circulating water at a given temperature through a stainless steel coil placed inside the dry fermentor unit. A sprayer was introduced under the cap of the dry fermentor to spray the ST liquid through the FVW in order to have sufficient moist for the microbes and also to washout the metabolites adhered on the FVW. A volumetric gas flow meter (mFlow, Bioprocess Control AB, Sweden), having detection range from 20 to 4000 NmL/h
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autoclave. The composition of the basal medium (per liter of distilled water) used contained 1.25 g/L NaHCO3; 0.5 g/L NH4Cl; 0.25 g/L KH2PO4; 0.25 g/L CaCl2, 0.032 g/L NiSO4; 0.32 g/L MgSO4$7H2O; 0.02 g/L FeCl2; 0.0144 g/L Na2MoO4$2H2O; 0.023 g/ L ZnCl2; 0.021 g/L CoCl2$6H2O; 0.01 g/L CuCl2$6H2O and 0.03 g/L MnCl2$4H2O. The working volume was then made up with FVW prehydrolyzates liquid fraction and the remaining volume with distilled water to reach a total volume of 8 L. The ST was then flushed for a few minutes with argon before starting the experiment to ensure anaerobic conditions inside the reactor. It was maintained at constant temperature of 55 ± 1 C with a constant agitation of 250 rpm and the pH of the ST was monitored regularly and was maintained between 5.5 and 6.75. The dry fermentor (DF) basket was either filled with solid fraction of the pre-treated or un-pre-treated FVW and was mixed with preheated inoculum and 40(X) basal medium. The basket was then placed inside the DF and the DF cap was closed airtight. Later, the DF was purged with argon for few minutes to create anaerobic conditions inside the reactor. During the feeding days, the Leachate (L) from the DF was drained through the leachate collection tube and the respective volume of liquid from the ST was removed and introduced into the DF. The collected Leachate (L) was then introduced into the ST. Argon was flushed through the DF once every other day to remove the headspace CO2.
Autoclaved and size <5 cm FVW (experiment I)
Fig. 1 e Dry fermentation experimental set-up: (a) stirred tank reactor (ST) and (b) dry fermentor.
(NmL ¼ normalized volume measurement at 0 Celsius and 1 standard atmosphere (atm), was connected at the gas outlet of the percolation tank and the dry fermentor in order to accurately record the total amount gas produced. The gas flowrate and total gas volume measured were normalized values with real-time pressure and temperature. Gas sampling ports were also connected to the gas outlets of both ST and DF for characterizing the gaseous products by using a gas chromatography.
Dry fermentation experiments Two independent dry fermentation experiments were performed using the following condition of FVW: (1) autoclaved FVW with size <5 cm (experiment I) and (2) raw FVW with size <5 cm (experiment II). For each experiment, the ST reactor was filled with 4.3 L sludge from the biogas plant along with 200 ml of 40 (X) times concentrated basal medium. Later, the ST reactor was heat treated at 105 C for 10 min using an
This experiment was performed with grinded (<5 cm) and autoclaved FVW, presumably favouring reduction in cellulose crystallinity while increasing the porosity of biomass; thereby making them susceptible to hydrolysis which would help to improve the overall hydrogen productivity from the process. 6.5 kg FVW, wet weight (ww), were first chopped and grinded using a grinder and they were autoclaved at 120 C and 15 psi (100 kPa) for 20 min. After autoclaving, around 2.1 L of liquid pre-hydrolyzate and 4.27 kg of solid fraction were separated. The collected liquid fraction mainly contained products from the pre-hydrolysis of sugars, starch and hemicelluloses, and was added into the ST which already contained 4.3 L of pre-treated sludge and 0.2 L of basal medium. In order to have a total working volume of 8 L in the ST, the rest of the volume was made up using distilled water. The separated solid fraction of FVW was then mixed with pre-treated sludge (0.61 L) along with basal medium inside the DF basket and it was placed inside the DF.
Non-autoclaved and size <5 cm FVW (experiment II) In this experiment FVW were chopped, grinded and then fed directly without any further pretreatment into the DF. The collected liquid fraction of 1.2 L after grinding the waste was added to ST that contained 4.3 L of pre-treated sludge and 0.2 ml basal medium. 2.5 L of distilled water was then added to have a final working volume of 8 L. The solid fraction was treated in the DF as explained above in Section Dry fermentation experiments. Each day, a gas sample of 3e5 mL was collected from the outlet sampling port of the ST and DF using a gastight Hamilton syringe. It was analysed on a gas chromatograph in order
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to monitor the concentrations of various gas components (H2, CO2 and CH4) present in the headspace of the reactors. Similarly, 10 mL of liquid samples from the ST and DF were withdrawn during the feeding days (alternative days) for measuring the metabolites concentrations, NHþ 4 , soluble COD (sCOD) and soluble sugar concentrations.
Analytical methods Gas-phase H2 concentrations were measured by 6890 N (Agilent technologies) gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) by injecting 3e5 mL of headspace sample collected using a gas tight syringe (Agilent). The GC was fitted with Hayesep D 80/100 packed column. The injector, oven and detector temperatures maintained at 150, 35 and 150 C, respectively. Argon was used as the carrier gas at a flow rate of 20 mL/min. The soluble metabolites were analysed by GC (6890 N Agilent) equipped with a flame ionization detector and TR-FFAP 30 m 0.25 mm ID 0.25 mm (Thermo Scientific). The initial temperature of the column was 40 C for 3 min followed with a ramp of 20 C/min to 60 C for 3 min and then increased at 30 C/min to 120 C for 4 min and reach a final temperature with ramp of 30 C/min to 240 C for 6 min. The temperatures of the injector and detector were both 240 C. Helium was used as the carrier gas at constant pressure of 103 kPa. The liquid sample was centrifuged and filtered using 0.22 mm nylon syringe filter and was acidified using orthophosphoric acid before analysing via GC. The soluble chemical oxygen demand (sCOD) was determined by using Hach LCK 114 COD kit and concentration was read directly using the Hach Pocket Colorimeter II. NHþ 4 concentration determination was performed using Spectroquant Ammonium Test kit and the absorbance was measured using Spectroquant photometer. The soluble carbohydrate concentrations were measured using phenol sulphuric acid method by Masuko et al. [27]. Total solids (TS) and volatile solids (VS) were also determined using standard methods [28].
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Results Two independent experiments were performed to understand the extent of influence on autoclaving the waste as a method for pretreatment for biohydrogen production under thermophilic condition. Biomass comprises of mainly cellulose, hemicellulose and lignin, in which, lignin forms a protective cover over cellulose and hemicellulose thus protecting the biomass from microbial attack [29]. However, cellulose and hemicellulose mainly polymers of simple sugars and can be useful for biofuel generation. In order to utilize these sugars, the lignin part has to be depolymerized/removed. In this respect, several pretreatment methods which are physical, chemical or biological has been applied to biomass either individually or in combination [30e32]. Identical FVW was used for both experiments and the FVW was first grinded in to pieces with size <5 cm. In one set of experiment (experiment I), grinded waste was further subjected to autoclaving at 120 C for 20 min at 15 psi (100 kPa). The liquid fraction (prehydrozylates) collected after autoclaving was treated in ST and solid fraction was treated in the DF using the heat treated inoculum that contains hydrogen producers.
Autoclaved and size <5 cm FVW (experiment I) The results of daily and cumulative hydrogen production (NmL) and percentage of H2 (%) at the headspace of both ST and DF were measured and is shown in Fig. 2. A cumulative total of 17,034 NmL of hydrogen was produced from this experiment with a yield of 27.19 NmL H2/gVS. While considering the daily production achieved throughout the experiment, it can be observed that maximum daily hydrogen production (9108 NmL) was achieved with in a day after the initiation of the fermentation. However, this high production rate decreased profoundly for the rest of the experimental days as can be observed from the Fig. 2, and that hydrogen obtained were 1907 and 3288 NmL respectively on second and third day, which is
Fig. 2 e Daily and cumulative hydrogen production, and H2% achieved for the experiment I.
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20.93 and 36.09% that produced in the first day. High hydrogen production obtained during the initial days of the experiment is due to the presence of high amount of simple sugars produced from autoclaving the waste and besides, the presence of active hydrogen producing bacteria in the inoculum that was achieved by pre-treating the sludge might also help to improve the hydrogen production from the process. It is worth noticing that about 84% of the total cumulative hydrogen was obtained within 3 days of the experimental run as this important in terms of economics of the process. The maximum hydrogen % achieved in the head space of DF and ST was, 41.08 and 27.3% respectively. One of major parameter that influences the batch fermentation is the effect of accumulated metabolites such as acids and alcohols [33]. There are reports that high concentration of these soluble metabolites may cause inhibitory effect to microbial consortium [34,35]. Major metabolites produced were acetic and iso-butyric acid and their maximum concentrations measured in the liquid phase were 7366 and 7662 mg/L, respectively. Varying concentrations of ethanol and butanol were also detected in the liquid phase with maximum concentrations reaching a value of 2510 and 427.2 mg/L, respectively (Fig. 3). Liquid phase concentrations of total sugar, ammonia and soluble COD were also monitored. The total sugar concentrations in ST on the start-up of the experiment were 19,515 mg/L which then reduced to 1000 mg/L within a day. However, this concentration got stabilized from 4th day with concentrations in ST and DF were ranging between 450 and 460 and 7000e9000 mg/L, respectively till the end of the experiment. Ammonia concentration is another influential factor that plays vital role in the performance and stability of the anaerobic digestion process [36]. Ammonia concentration is greatly influenced by C/N ratio and thus affects the outcome of the process [37]. The concentrations measured in the liquid phase where in the range between 490 and 700 mg/L in DF and 1950e2200 mg/L in ST.
Non-autoclaved and size <5 cm FVW (experiment II) In this experiment, FVW was grinded and added directly into the DF. The cumulative hydrogen gas production reached
10,761 NmL on the third day after the fermentation started which is about 82.5% of the total hydrogen produced from the experiment. The hydrogen yield from the process was 20.81 NmL H2/gVS. Also, comparing the daily hydrogen production with the autoclaved waste, maximum hydrogen production was obtained after three days of the process with production of 6684 NmL (Fig. 4). This can be considered as an effect of high temperature fermentation condition used in this study which causes the pre-hydrolysis of the waste generating simple sugars. The maximum percentage of hydrogen measured at the head space was 19.63 and 20.89%, respectively in ST and DF. The concentrations of VFA measured in the liquid fraction were mainly contained acetic acid and iso-butyric acid with maximum of 10,535 and 10,848 mg/L, respectively (Fig. 5). This is about 42e43% increase in both acetic acid and iso-butyric acid production compared to experiment I. The total sugar concentration in the liquid fraction was also measured periodically and the maximum concentration of 18,436 mg/L was obtained on the second day from the DF. This result is in accordance with the higher amount of hydrogen produced (5280 NmL) on the third day in the ST as leachate collected from DF on the second day was fed as substrate in ST. The ammonia concentration in DF leachate was between 550 and 1050 mg/L and that of liquid from ST were in between the range of 1600e2100 mg/L during the course of the experiment.
Discussion Recently much interest has been turned towards the use of waste to generate platform chemicals and energy to alleviate the dependency on fossil fuels along with reducing the greenhouse gas emission to the atmosphere [38,39]. Dark fermentation is one such technique that facilitates the waste treatment of various organic substrates such as wastewater, organic fraction of municipal solid wastes, etc. with simultaneous resource recovery in the form of various platform chemicals C2 to C7 and hydrogen production. Most dark fermentation studies were mainly performed in wet (liquid) mode to digest and recover energy from these organic matters
Fig. 3 e Volatile fatty acid (VFA) production profile in experiment I.
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Fig. 4 e Daily and cumulative hydrogen production, and H2% achieved for the experiment II.
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Concentrations (mg/L)
20000 Acetic acid vs Days ISobut vs Days Total VFA vs Days
15000
10000
5000
0 0
1
2
3
4
5
6
7
Time (days)
Fig. 5 e Volatile fatty acid (VFA) production profile in experiment II.
present either in wastewater or by diluting the solid waste. In contract, dry fermentation is relatively new approach which operates at higher total solid content with less use of water. This fermentation type is well established in Europe to generate biogas from lignocellulosic waste and organic faction of municipal solid waste. Recently, dry fermentation studies were being carried out for biohydrogen production and to improve the productivity of the process. One such studies were performed by Elsamadony and Tawfik [40] where codigesting OFMSW with protein and calcium-rich substrates such as gelatin solid waste (GSW) and paper mill sludge (PMS) were carried out, and were observed that a combination of 70% OFMSW þ 20% GSW þ 10% PM resulted in improving the hydrogen yield and production. Positive effects of co-digestion were also observed by Tyagi et al. [41] on mixing OFMSW with
sewage sludge. Elsamadony and Tawfik research group has also studied the effect of various additives such as surfactant Tween 80 and polyethylene glycol [42], and buffering agent such as sodium bicarbonate on improving the biohydrogen production [43]. Accumulation of high concentrations of VFA and ammonia results from the high organic loading rate in dry fermentation is always detrimental to microbial community. As an effort to optimize and stabilize the dry fermentation system for biohydrogen generation various strategies have been adopted in this study as follows. One such approach was the installation of a feeding tank (ST) with active hydrogen producers side by side to the DF for periodically feeding microbes to the DF as well as to wash out the accumulated VFAs that can cause pH drop in DF. In addition, spraying inoculum over DF
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intermittently will allow the DF system to attain more buffering capacity. One of the important parameter that affect the stability of DF is the Feed/Inoculum (F/I) ratio. Higher inoculum addition initially will provide better start-up condition as more microbes available for the biological process [44]. However, using large amount of inoculum (low F/I ratio) will result in faster VFA production and eventually leads to instability of system. To overcome this situation, in this study we used a high F/I ratio of 6.98 (wet basis); and the leachate collection and ST liquid feeding frequencies were further optimized to improve the hydrogen yield. In thermophilic condition, high F/I ratio is reported to yield more biogas than low F/I ratio tested [45]. As agitation was completely devoid in our system, in order to ensure balance in distribution of microbes and nutrients, complete premixing of the inoculum with the feed was done before introduce into the DF system. Leachate recirculation is important in DF as it helps to redistribute the nutrients and microorganisms [46]. In addition, removal of accumulated hydrogen from the headspace of the DF was also done to prevent the consumption of produced hydrogen by homoacetogens [47,48]. Another operating parameter tested were the temperature which in this study was maintained at constant temperature of 55 C. Compared to mesophilic condition, thermophilic condition allows faster hydrolysis, which is considered as the rate limiting step in dark fermentation [23]. This is in accordance with the result we obtained with our previous studies performed under mesophilic condition (under revision). As expected, experiments using autoclaved waste produced more hydrogen than without autoclaving as it favours solubilisation of complex waste biomass. However, an enhancement in overall hydrogen production was observed under thermophilic (autoclaved or non-autoclaved) condition tested in this study than in our previous studies tested under mesophilic conditions. It was also found that pretreated (autoclaved) waste (experiment I) generated 30% higher hydrogen compared to the non-autoclaved one (experiment II). It can be observed from Figs. 2 and 4 that daily hydrogen production was higher on third day compared to the second day. This can be attributed to the effect of higher fermentation temperature employed in this process that might facilitate the improvement in hydrolysis of the waste with time and thereby enhancing the biohydrogen production. The hydrogen yields obtained in this study were similar to that obtained by Aguilar et al. [49]. They obtained a yield of 24.3 NmL H2/gVS with a continuous stirred tank reactor at thermophilic dry condition treating OFMSW. Several pretreatment methods have been tested for wet fermentation for biogas and biohydrogen production. Although, dry fermentation technology for biogas generation have been tested in full scale only few pretreatment methods have been investigated so far. As hydrogen production by dry fermentation is relatively new techniques, in this study, the effect of autoclaving as a pretreatment method were tested. Although relatively low temperature and pressure to the waste were applied through autoclaving (121 C and at 100 kPa for 20 min) compared to the common liquid hot water pretreatment, these approaches offer several advantages over other physicochemical pretreatment methods such as generation of less or no inhibitory compounds and non-requirement of
neutralization before fermentation and were applied [50]. The presence of ethanol and propionic acid were also noticed in the DF and ST which might be another reason as metabolic pathway of former does not leads to hydrogen production, whereas, the latter is a hydrogen consuming pathway [23].
Conclusions Dry anaerobic fermentation of FVW for biohydrogen generation was successfully performed in a 55-L reactor. From the studies, experiment I using autoclaved waste was found to generate a cumulative total of 17,033 NmL H2 (27.19 NmL H2/gVS) and 13,041 NmL H2 (20.81 NmL H2/gVS) in experiment II. VFAs are profoundly produced in both experiments (Experiment I, maximum total VFAs ¼ 17,432 mg/L and experiment II, maximum total VFAs ¼ 23,448 mg/L) which could influence the overall performance of the process. It can be concluded that although thermophilic autoclaved improved the overall hydrogen productivity, further optimization studies are warranted for commercialization of this process.
Acknowledgements The authors wish to thank TUBITAK-MAG-215 M 314 for financial support of this study. HNA thanks the Xunta de Galicia (Spain) for his postdoctoral fellowship (ED 481B-2016/ 195-0). The authors Bensu Gu¨nay and Okyanus Yazgın would also like to thank the TUBITAK 2209-A University Student Research Projects Support Program for this research.
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L, Pauss A, Ribeiro T. Solid anaerobic digestion: state[26] Andre of-art, scientific and technological hurdles. Bioresour Technol 2018;247:1027e37. [27] Masuko T, Minami A, Iwasaki N, Majima T, Nishimura SI, Lee YC. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 2005;339:69e72. [28] APHA/AWWA/WEF. Standard methods for the examination of water and wastewater. Stand Methods 2012;541. [29] Chong ML, Sabaratnam V, Shirai Y, Hassan MA. Review: biohydrogen production from biomass and industrial wastes by dark fermentation. Int J Hydrogen Energy 2009;34:3277e87. [30] Urbaniec K, Bakker RR. Biomass residues as raw material for dark hydrogen fermentation- a review. Int J Hydrogen Energy 2015;40:3648e58. [31] Bundhoo MAZ, Mohee R, Hassan MA. Effects of pretreatment technologies on dark fermentative biohydrogen production: a review. J Environ Manag 2015;157:20e48. [32] Rafieenia R, Lavagnolo MC, Pivato A. Pre-treatment technologies for dark fermentative hydrogen production: current advances and future directions. Waste Manag 2018;71:734e48. [33] Van Ginkel S, Logan BE. Inhibition of biohydrogen production by undissociated acetic and butyric acids. Environ Sci Technol 2005;39:9351e6. [34] Bundhoo MAZ, Mohee R. Inhibition of dark fermentative bio-hydrogen production: a review. Int J Hydrogen Energy 2016;41(16):6713e33. [35] Elbeshbishy E, Dhar BR, Nakhla G, Lee HS. A critical review on inhibition of dark biohydrogen fermentation. Renew Sustain Energy Rev 2017;79:656e68. [36] Salerno MB, Park W, Zuo Y, Bruce EL. Inhibition of biohydrogen production by ammonia. Water Res 2006;40:1167e72. [37] Wu S, Shen Z, Yang C, Zhou Y, Li X, Zeng G, et al. Effects of C/N ratio and bulking agent on speciation of Zn and Cu and enzymatic activity during pig manure composting. Int Biodeterior Biodegrad 2017;119:429e36. [38] Xu F, Li Y, Ge X, Yang L, Li Y. Anaerobic digestion of food waste e challenges and opportunities. Bioresour Technol 2018;247:1047e58. [39] Kennes D, Abubackar HN, Diaz M, Veiga MC, Kennes C. Bioethanol production from biomass: carbohydrate vs syngas fermentation. J Chem Technol Biotechnol 2016;91(2):304e17. [40] Elsamadony M, Tawfik A. Dry anaerobic co-digestion of organic fraction of municipal waste with paperboard mill sludge and gelatin solid waste for enhancement of hydrogen production. Bioresour Technol 2015;191:157e65. [41] Tyagi VK, Angeriz Campoy R, Alvarez-Gallego CJ, Romero Garcia LI. Enhancement in hydrogen production by thermophilic anaerobic co-digestion of organic fraction of municipal solid waste and sewage sludgeeoptimization of treatment conditions. Bioresour Technol 2014;164:408e15. [42] Elsamadony M, Tawfik A. Surfactant-enhanced biohydrogen production from organic fraction of municipal solid waste via dry anaerobic digestion. Appl Energy 2015;149:272e82. [43] Elsamadony M, Tawfik A. Potential of biohydrogen production from organic fraction of municipal solid waste (OFMSW) using pilot-scale dry anaerobic reactor. Bioresour Technol 2015;196:9e16. [44] Yan Z, Song Z, Li D, Yuan Y, Liu X, Zheng T. The effects of initial substrate concentration, C/N ratio, and temperature on solid-state anaerobic digestion from composting rice straw. Bioresour Technol 2015;177:266e73.
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Biohydrogen production from autoclaved fruit and vegetable wastes by dry fermentation under thermophilic condition Haris Nalakath Abubackar a,b, Tugba Keskin a, Okyanus Yazgin a, Bensu Gunay a, Kubra Arslan a, Nuri Azbar a,* a
Bioengineering Department, Faculty of Engineering, Ege University, 35100, Izmir, Turkey Chemical Engineering Laboratory, Faculty of Sciences and Centre for Advanced Scientific Research (CICA), ~ a, Ru´a da Fraga 10, E e 15008 A Corun ~ a, Spain University of La Corun
b
article info
abstract
Article history:
Dark fermentative hydrogen production from organic waste is an attractive technique that
Received 15 October 2018
simultaneously treats waste along with generation of renewable fuel. In this study, a
Received in revised form
relative new technology named dark dry fermentation was tested in a 55-L reactor to treat
29 November 2018
fruit and vegetable waste (FVW) along with simultaneous generation of biohydrogen. To
Accepted 8 December 2018
understand the effect of autoclaving as a pretreatment method on FVW for subsequent
Available online 8 January 2019
biohydrogen production, two independent experiments were performed; one with autoclaved waste (experiment I) and another by using non autoclaved waste (experiment II).
Keywords:
From the analyses, it was found that maximum hydrogen % obtained for experiment I was
Biohydrogen
41% (v/v%) whereas, for experiment II was 21%. In terms of total hydrogen produced,
Autoclaving
around 30% higher production was observed with experiment I compared to experiment II.
Fruit and vegetable waste
The hydrogen yields for experiment I and experiment II were respectively, 27.19 and 20.81
Bioenergy
NmL H2/gVS (VS ¼ volatile solid added), and the metabolites (VFAs) preferentially produced were acetic acid and iso-butyric acid. © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction The worldwide generation of organic fraction of municipal solid waste (OFMSW) is estimated to be around 1012 million metric tons per year by 2025 [1]. These biodegradable wastes are generally disposed by landfilling or by open dumping. One way to treat them properly together with simultaneous generation of energy is by applying anaerobic digestion [2]. This not only mitigates the climate change caused from the greenhouse gas emissions but also helps to achieve
sustainability in terms of energy. Several countries set targets to reduce the emission of environmentally harmful gases. In this respect, European Environment Agency target to achieve a reduction of 40% greenhouse gas emissions cuts by 2030. According to the revised European Directive 2009/28/EC, by 2030, 27% renewable energy consumption should be achieved. Overall, to meet these requirements, a huge leap in research has been noticed for the past decade to find an alternative renewable energy source [3,4]. Hydrogen is considered as a promising energy source in terms of its high energy content (143 MJ/kg) compared to any
* Corresponding author. E-mail address: [email protected] (N. Azbar). https://doi.org/10.1016/j.ijhydene.2018.12.068 0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
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existing fuel, and its combustion generates water as the only by-product. To date, hydrogen was produced mainly from fossil fuels with 48% from natural gas, 30% from heavy oils and naphtha, 18% from coal, and 4% from electrolysis [5]. Recently hydrogen gas generation from renewable sources such as biomass, which is organic materials derived from plants and animals, is gaining much attention [6]. Biomass can be thermo-chemically or biologically treated to generate hydrogen where former mainly involves pyrolysis or gasification and catalytic steam reforming. Biological technologies include direct and indirect biophotolysis using algae and cyanobacteria, photo-fermentation by photosynthetic bacteria and dark fermentation by fermentative bacteria, and microbial electrolysis cell [7,8]. Dark fermentative bacteria use carbohydrate rich substrates and oxidise them releasing electrons, which under anaerobic or anoxic, dark conditions accepted by protons and thereby generating molecular H2. This is accompanied by the formation of various organic acids [9]. As a model substrate, glucose, upon fermentation produces mainly acetic acid and butyric acid along with a theoretical yield of 4 and 2 mol of H2, respectively [10]. It is considered as a promising technology in terms of its practicality to utilize different types of organic wastes as feedstocks and thus can also able to improve the economics of the process. Potential biomass waste for biohydrogen includes agricultural residue, industrial residue, livestock waste, OFMSW [11,12]. These lignocellulosic materials are recalcitrant for biodegradation unless suitable pretreatment methods have been applied. Several pretreatment methods have been developed and their effects on biogas and biohydrogen production were evaluated during the past decades [13,14]. Autoclaving is considered to be an ideal method as it offers several advantages over other physicochemical methods such as requirement of no chemicals and could able to preserve the nutrition present in the waste [15]. An ideal feedstock for biohydrogen should be rich in carbohydrate and deficient in nitrogen [16]. Fruit and vegetable wastes (FVW), which accounts for 45% of the human food lost, is an ideal candidate for fermentation in terms of its moisture content, biodegradability, low total solid and high volatile solid content [17]. Several studies have been undergone using fruit and vegetables by anaerobic digestion for biogas production [2,18] and by dark fermentation for biohydrogen production [19e21]. However, due the complexity of the structure, microorganisms are less access to the biodegradable substrate causing a reduction in the overall productivity from the process. Thus, waste has to be subjected to pretreatment for efficient cellulose digestion [22]. The process temperature is one of the main parameters that influence the biohydrogen production by dark fermentation. Operating under thermophilic condition (42e75 C) has several advantages over mesophilic condition including the inhibition of the hydrogen consuming microorganisms and possibility to treat directly the hot industrial effluents [10,23]. Anaerobic digestion can be classified into: wet fermentation where the total solid content is less than 10% and dry fermentation with total solid content greater than 10%. Dry fermentation is a promising technology due to its several advantages like requirement of smaller reactor volume, easiness in handling digestate and lesser water wastage [24e26].
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Although many studies have performed for the biogas production through dry fermentation still not much studies on biohydrogen production using this technology. In this study, dry fermentation studies were performed under thermophilic condition to understand the effect of pretreatment (autoclaving) of FVW on biohydrogen production. To the best of authors’ knowledge this study is a novel in terms of combining various factors such as a pretreatment applied, substrate utilized and fermentation temperature in dry fermentation system.
Experimental section Feedstock and inoculum The FVW was obtained from the Izmir municipality bazar (Turkey) and were either chopped into very small pieces using a grinder or cut into small pieces of size < 5 cm. The composition of FVW collected, expressed in mass percentage (wt%), were: radish (5.71), pepper (5.06), pomegranate (7.02), pear (3.33), apple (4.66), pumpkin (2.06), mandarin (1.68), tomato (7.45), onion (6.20), potato (5.48), peach (12.90), lemon (2.28), eggplant (11.34), carrot (8.68), orange (2.59), cucumber (6.70), cabbage (4.19), grape (2.68). The same FVW composition was used for all the experimental studies conducted in dry fermentor (DF) and reported in this manuscript. The inoculum was collected from a local biogas plant that uses dairy products and local animal wastes as feed (Izmir, Turkey) and underwent a pre-treatment at 105 C for 10 min in order to gather hydrogen producers. The characteristics of the pretreated inoculum used in these studies were: pH 9.45, soluble COD of 9000 mg/L, total sugar of 900 mg/L, ammonium concentration of 3610 mg/L, TS (total solid) and VS of 7.9 and 4.8%, respectively.
Dry fermentation system The experiments were performed in a fully automated stirred tank (ST) single walled reactor of a total volume of 13 L (INFORS HT Labfors 5 reactor, Switzerland) which contained hydrogen producing microbes and a 55-L custom-made reactor (DF) where the FVW and hydrogen producers were mixed and filled for treatment (Fig. 1). Temperature of the ST reactor was controlled via an inbuilt platinum resistor temperature sensor (pt100) that was inserted in a blink pocket of the reactor and by using cold finger to circulate the cooling water. The dry fermentor was fabricated from an autoclave unit and an aluminum basket with holes around the sides and bottoms placed inside the unit for introducing the FVW and holding it, and allowing the leachate to flow freely to the bottom of the dry fermentor unit to the leachate collection tube. The inside temperature of the dry fermentor unit was controlled by circulating water at a given temperature through a stainless steel coil placed inside the dry fermentor unit. A sprayer was introduced under the cap of the dry fermentor to spray the ST liquid through the FVW in order to have sufficient moist for the microbes and also to washout the metabolites adhered on the FVW. A volumetric gas flow meter (mFlow, Bioprocess Control AB, Sweden), having detection range from 20 to 4000 NmL/h
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autoclave. The composition of the basal medium (per liter of distilled water) used contained 1.25 g/L NaHCO3; 0.5 g/L NH4Cl; 0.25 g/L KH2PO4; 0.25 g/L CaCl2, 0.032 g/L NiSO4; 0.32 g/L MgSO4$7H2O; 0.02 g/L FeCl2; 0.0144 g/L Na2MoO4$2H2O; 0.023 g/ L ZnCl2; 0.021 g/L CoCl2$6H2O; 0.01 g/L CuCl2$6H2O and 0.03 g/L MnCl2$4H2O. The working volume was then made up with FVW prehydrolyzates liquid fraction and the remaining volume with distilled water to reach a total volume of 8 L. The ST was then flushed for a few minutes with argon before starting the experiment to ensure anaerobic conditions inside the reactor. It was maintained at constant temperature of 55 ± 1 C with a constant agitation of 250 rpm and the pH of the ST was monitored regularly and was maintained between 5.5 and 6.75. The dry fermentor (DF) basket was either filled with solid fraction of the pre-treated or un-pre-treated FVW and was mixed with preheated inoculum and 40(X) basal medium. The basket was then placed inside the DF and the DF cap was closed airtight. Later, the DF was purged with argon for few minutes to create anaerobic conditions inside the reactor. During the feeding days, the Leachate (L) from the DF was drained through the leachate collection tube and the respective volume of liquid from the ST was removed and introduced into the DF. The collected Leachate (L) was then introduced into the ST. Argon was flushed through the DF once every other day to remove the headspace CO2.
Autoclaved and size <5 cm FVW (experiment I)
Fig. 1 e Dry fermentation experimental set-up: (a) stirred tank reactor (ST) and (b) dry fermentor.
(NmL ¼ normalized volume measurement at 0 Celsius and 1 standard atmosphere (atm), was connected at the gas outlet of the percolation tank and the dry fermentor in order to accurately record the total amount gas produced. The gas flowrate and total gas volume measured were normalized values with real-time pressure and temperature. Gas sampling ports were also connected to the gas outlets of both ST and DF for characterizing the gaseous products by using a gas chromatography.
Dry fermentation experiments Two independent dry fermentation experiments were performed using the following condition of FVW: (1) autoclaved FVW with size <5 cm (experiment I) and (2) raw FVW with size <5 cm (experiment II). For each experiment, the ST reactor was filled with 4.3 L sludge from the biogas plant along with 200 ml of 40 (X) times concentrated basal medium. Later, the ST reactor was heat treated at 105 C for 10 min using an
This experiment was performed with grinded (<5 cm) and autoclaved FVW, presumably favouring reduction in cellulose crystallinity while increasing the porosity of biomass; thereby making them susceptible to hydrolysis which would help to improve the overall hydrogen productivity from the process. 6.5 kg FVW, wet weight (ww), were first chopped and grinded using a grinder and they were autoclaved at 120 C and 15 psi (100 kPa) for 20 min. After autoclaving, around 2.1 L of liquid pre-hydrolyzate and 4.27 kg of solid fraction were separated. The collected liquid fraction mainly contained products from the pre-hydrolysis of sugars, starch and hemicelluloses, and was added into the ST which already contained 4.3 L of pre-treated sludge and 0.2 L of basal medium. In order to have a total working volume of 8 L in the ST, the rest of the volume was made up using distilled water. The separated solid fraction of FVW was then mixed with pre-treated sludge (0.61 L) along with basal medium inside the DF basket and it was placed inside the DF.
Non-autoclaved and size <5 cm FVW (experiment II) In this experiment FVW were chopped, grinded and then fed directly without any further pretreatment into the DF. The collected liquid fraction of 1.2 L after grinding the waste was added to ST that contained 4.3 L of pre-treated sludge and 0.2 ml basal medium. 2.5 L of distilled water was then added to have a final working volume of 8 L. The solid fraction was treated in the DF as explained above in Section Dry fermentation experiments. Each day, a gas sample of 3e5 mL was collected from the outlet sampling port of the ST and DF using a gastight Hamilton syringe. It was analysed on a gas chromatograph in order
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to monitor the concentrations of various gas components (H2, CO2 and CH4) present in the headspace of the reactors. Similarly, 10 mL of liquid samples from the ST and DF were withdrawn during the feeding days (alternative days) for measuring the metabolites concentrations, NHþ 4 , soluble COD (sCOD) and soluble sugar concentrations.
Analytical methods Gas-phase H2 concentrations were measured by 6890 N (Agilent technologies) gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) by injecting 3e5 mL of headspace sample collected using a gas tight syringe (Agilent). The GC was fitted with Hayesep D 80/100 packed column. The injector, oven and detector temperatures maintained at 150, 35 and 150 C, respectively. Argon was used as the carrier gas at a flow rate of 20 mL/min. The soluble metabolites were analysed by GC (6890 N Agilent) equipped with a flame ionization detector and TR-FFAP 30 m 0.25 mm ID 0.25 mm (Thermo Scientific). The initial temperature of the column was 40 C for 3 min followed with a ramp of 20 C/min to 60 C for 3 min and then increased at 30 C/min to 120 C for 4 min and reach a final temperature with ramp of 30 C/min to 240 C for 6 min. The temperatures of the injector and detector were both 240 C. Helium was used as the carrier gas at constant pressure of 103 kPa. The liquid sample was centrifuged and filtered using 0.22 mm nylon syringe filter and was acidified using orthophosphoric acid before analysing via GC. The soluble chemical oxygen demand (sCOD) was determined by using Hach LCK 114 COD kit and concentration was read directly using the Hach Pocket Colorimeter II. NHþ 4 concentration determination was performed using Spectroquant Ammonium Test kit and the absorbance was measured using Spectroquant photometer. The soluble carbohydrate concentrations were measured using phenol sulphuric acid method by Masuko et al. [27]. Total solids (TS) and volatile solids (VS) were also determined using standard methods [28].
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Results Two independent experiments were performed to understand the extent of influence on autoclaving the waste as a method for pretreatment for biohydrogen production under thermophilic condition. Biomass comprises of mainly cellulose, hemicellulose and lignin, in which, lignin forms a protective cover over cellulose and hemicellulose thus protecting the biomass from microbial attack [29]. However, cellulose and hemicellulose mainly polymers of simple sugars and can be useful for biofuel generation. In order to utilize these sugars, the lignin part has to be depolymerized/removed. In this respect, several pretreatment methods which are physical, chemical or biological has been applied to biomass either individually or in combination [30e32]. Identical FVW was used for both experiments and the FVW was first grinded in to pieces with size <5 cm. In one set of experiment (experiment I), grinded waste was further subjected to autoclaving at 120 C for 20 min at 15 psi (100 kPa). The liquid fraction (prehydrozylates) collected after autoclaving was treated in ST and solid fraction was treated in the DF using the heat treated inoculum that contains hydrogen producers.
Autoclaved and size <5 cm FVW (experiment I) The results of daily and cumulative hydrogen production (NmL) and percentage of H2 (%) at the headspace of both ST and DF were measured and is shown in Fig. 2. A cumulative total of 17,034 NmL of hydrogen was produced from this experiment with a yield of 27.19 NmL H2/gVS. While considering the daily production achieved throughout the experiment, it can be observed that maximum daily hydrogen production (9108 NmL) was achieved with in a day after the initiation of the fermentation. However, this high production rate decreased profoundly for the rest of the experimental days as can be observed from the Fig. 2, and that hydrogen obtained were 1907 and 3288 NmL respectively on second and third day, which is
Fig. 2 e Daily and cumulative hydrogen production, and H2% achieved for the experiment I.
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20.93 and 36.09% that produced in the first day. High hydrogen production obtained during the initial days of the experiment is due to the presence of high amount of simple sugars produced from autoclaving the waste and besides, the presence of active hydrogen producing bacteria in the inoculum that was achieved by pre-treating the sludge might also help to improve the hydrogen production from the process. It is worth noticing that about 84% of the total cumulative hydrogen was obtained within 3 days of the experimental run as this important in terms of economics of the process. The maximum hydrogen % achieved in the head space of DF and ST was, 41.08 and 27.3% respectively. One of major parameter that influences the batch fermentation is the effect of accumulated metabolites such as acids and alcohols [33]. There are reports that high concentration of these soluble metabolites may cause inhibitory effect to microbial consortium [34,35]. Major metabolites produced were acetic and iso-butyric acid and their maximum concentrations measured in the liquid phase were 7366 and 7662 mg/L, respectively. Varying concentrations of ethanol and butanol were also detected in the liquid phase with maximum concentrations reaching a value of 2510 and 427.2 mg/L, respectively (Fig. 3). Liquid phase concentrations of total sugar, ammonia and soluble COD were also monitored. The total sugar concentrations in ST on the start-up of the experiment were 19,515 mg/L which then reduced to 1000 mg/L within a day. However, this concentration got stabilized from 4th day with concentrations in ST and DF were ranging between 450 and 460 and 7000e9000 mg/L, respectively till the end of the experiment. Ammonia concentration is another influential factor that plays vital role in the performance and stability of the anaerobic digestion process [36]. Ammonia concentration is greatly influenced by C/N ratio and thus affects the outcome of the process [37]. The concentrations measured in the liquid phase where in the range between 490 and 700 mg/L in DF and 1950e2200 mg/L in ST.
Non-autoclaved and size <5 cm FVW (experiment II) In this experiment, FVW was grinded and added directly into the DF. The cumulative hydrogen gas production reached
10,761 NmL on the third day after the fermentation started which is about 82.5% of the total hydrogen produced from the experiment. The hydrogen yield from the process was 20.81 NmL H2/gVS. Also, comparing the daily hydrogen production with the autoclaved waste, maximum hydrogen production was obtained after three days of the process with production of 6684 NmL (Fig. 4). This can be considered as an effect of high temperature fermentation condition used in this study which causes the pre-hydrolysis of the waste generating simple sugars. The maximum percentage of hydrogen measured at the head space was 19.63 and 20.89%, respectively in ST and DF. The concentrations of VFA measured in the liquid fraction were mainly contained acetic acid and iso-butyric acid with maximum of 10,535 and 10,848 mg/L, respectively (Fig. 5). This is about 42e43% increase in both acetic acid and iso-butyric acid production compared to experiment I. The total sugar concentration in the liquid fraction was also measured periodically and the maximum concentration of 18,436 mg/L was obtained on the second day from the DF. This result is in accordance with the higher amount of hydrogen produced (5280 NmL) on the third day in the ST as leachate collected from DF on the second day was fed as substrate in ST. The ammonia concentration in DF leachate was between 550 and 1050 mg/L and that of liquid from ST were in between the range of 1600e2100 mg/L during the course of the experiment.
Discussion Recently much interest has been turned towards the use of waste to generate platform chemicals and energy to alleviate the dependency on fossil fuels along with reducing the greenhouse gas emission to the atmosphere [38,39]. Dark fermentation is one such technique that facilitates the waste treatment of various organic substrates such as wastewater, organic fraction of municipal solid wastes, etc. with simultaneous resource recovery in the form of various platform chemicals C2 to C7 and hydrogen production. Most dark fermentation studies were mainly performed in wet (liquid) mode to digest and recover energy from these organic matters
Fig. 3 e Volatile fatty acid (VFA) production profile in experiment I.
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Fig. 4 e Daily and cumulative hydrogen production, and H2% achieved for the experiment II.
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Concentrations (mg/L)
20000 Acetic acid vs Days ISobut vs Days Total VFA vs Days
15000
10000
5000
0 0
1
2
3
4
5
6
7
Time (days)
Fig. 5 e Volatile fatty acid (VFA) production profile in experiment II.
present either in wastewater or by diluting the solid waste. In contract, dry fermentation is relatively new approach which operates at higher total solid content with less use of water. This fermentation type is well established in Europe to generate biogas from lignocellulosic waste and organic faction of municipal solid waste. Recently, dry fermentation studies were being carried out for biohydrogen production and to improve the productivity of the process. One such studies were performed by Elsamadony and Tawfik [40] where codigesting OFMSW with protein and calcium-rich substrates such as gelatin solid waste (GSW) and paper mill sludge (PMS) were carried out, and were observed that a combination of 70% OFMSW þ 20% GSW þ 10% PM resulted in improving the hydrogen yield and production. Positive effects of co-digestion were also observed by Tyagi et al. [41] on mixing OFMSW with
sewage sludge. Elsamadony and Tawfik research group has also studied the effect of various additives such as surfactant Tween 80 and polyethylene glycol [42], and buffering agent such as sodium bicarbonate on improving the biohydrogen production [43]. Accumulation of high concentrations of VFA and ammonia results from the high organic loading rate in dry fermentation is always detrimental to microbial community. As an effort to optimize and stabilize the dry fermentation system for biohydrogen generation various strategies have been adopted in this study as follows. One such approach was the installation of a feeding tank (ST) with active hydrogen producers side by side to the DF for periodically feeding microbes to the DF as well as to wash out the accumulated VFAs that can cause pH drop in DF. In addition, spraying inoculum over DF
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intermittently will allow the DF system to attain more buffering capacity. One of the important parameter that affect the stability of DF is the Feed/Inoculum (F/I) ratio. Higher inoculum addition initially will provide better start-up condition as more microbes available for the biological process [44]. However, using large amount of inoculum (low F/I ratio) will result in faster VFA production and eventually leads to instability of system. To overcome this situation, in this study we used a high F/I ratio of 6.98 (wet basis); and the leachate collection and ST liquid feeding frequencies were further optimized to improve the hydrogen yield. In thermophilic condition, high F/I ratio is reported to yield more biogas than low F/I ratio tested [45]. As agitation was completely devoid in our system, in order to ensure balance in distribution of microbes and nutrients, complete premixing of the inoculum with the feed was done before introduce into the DF system. Leachate recirculation is important in DF as it helps to redistribute the nutrients and microorganisms [46]. In addition, removal of accumulated hydrogen from the headspace of the DF was also done to prevent the consumption of produced hydrogen by homoacetogens [47,48]. Another operating parameter tested were the temperature which in this study was maintained at constant temperature of 55 C. Compared to mesophilic condition, thermophilic condition allows faster hydrolysis, which is considered as the rate limiting step in dark fermentation [23]. This is in accordance with the result we obtained with our previous studies performed under mesophilic condition (under revision). As expected, experiments using autoclaved waste produced more hydrogen than without autoclaving as it favours solubilisation of complex waste biomass. However, an enhancement in overall hydrogen production was observed under thermophilic (autoclaved or non-autoclaved) condition tested in this study than in our previous studies tested under mesophilic conditions. It was also found that pretreated (autoclaved) waste (experiment I) generated 30% higher hydrogen compared to the non-autoclaved one (experiment II). It can be observed from Figs. 2 and 4 that daily hydrogen production was higher on third day compared to the second day. This can be attributed to the effect of higher fermentation temperature employed in this process that might facilitate the improvement in hydrolysis of the waste with time and thereby enhancing the biohydrogen production. The hydrogen yields obtained in this study were similar to that obtained by Aguilar et al. [49]. They obtained a yield of 24.3 NmL H2/gVS with a continuous stirred tank reactor at thermophilic dry condition treating OFMSW. Several pretreatment methods have been tested for wet fermentation for biogas and biohydrogen production. Although, dry fermentation technology for biogas generation have been tested in full scale only few pretreatment methods have been investigated so far. As hydrogen production by dry fermentation is relatively new techniques, in this study, the effect of autoclaving as a pretreatment method were tested. Although relatively low temperature and pressure to the waste were applied through autoclaving (121 C and at 100 kPa for 20 min) compared to the common liquid hot water pretreatment, these approaches offer several advantages over other physicochemical pretreatment methods such as generation of less or no inhibitory compounds and non-requirement of
neutralization before fermentation and were applied [50]. The presence of ethanol and propionic acid were also noticed in the DF and ST which might be another reason as metabolic pathway of former does not leads to hydrogen production, whereas, the latter is a hydrogen consuming pathway [23].
Conclusions Dry anaerobic fermentation of FVW for biohydrogen generation was successfully performed in a 55-L reactor. From the studies, experiment I using autoclaved waste was found to generate a cumulative total of 17,033 NmL H2 (27.19 NmL H2/gVS) and 13,041 NmL H2 (20.81 NmL H2/gVS) in experiment II. VFAs are profoundly produced in both experiments (Experiment I, maximum total VFAs ¼ 17,432 mg/L and experiment II, maximum total VFAs ¼ 23,448 mg/L) which could influence the overall performance of the process. It can be concluded that although thermophilic autoclaved improved the overall hydrogen productivity, further optimization studies are warranted for commercialization of this process.
Acknowledgements The authors wish to thank TUBITAK-MAG-215 M 314 for financial support of this study. HNA thanks the Xunta de Galicia (Spain) for his postdoctoral fellowship (ED 481B-2016/ 195-0). The authors Bensu Gu¨nay and Okyanus Yazgın would also like to thank the TUBITAK 2209-A University Student Research Projects Support Program for this research.
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