Bioresource Technology 182 (2015) 103–113

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Acidogenic fermentation of food waste for volatile fatty acid production with co-generation of biohydrogen Shikha Dahiya, Omprakash Sarkar, Y.V. Swamy, S. Venkata Mohan ⇑ Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 VFA production from food waste

fermentation at variable redox conditions.  Short chain carboxylic acids synthesis was higher at alkaline pH.  Higher H2 production was also observed at alkaline pH.

a r t i c l e

i n f o

Article history: Received 16 October 2014 Received in revised form 29 December 2014 Accepted 3 January 2015 Available online 20 January 2015 Keywords: Degree of acidification Short chain carboxylic acids Volatile fatty acids (VFA) platform pH Buffering capacity

a b s t r a c t Fermentation experiments were designed to elucidate the functional role of the redox microenvironment on volatile fatty acid (VFA, short chain carboxylic acid) production and co-generation of biohydrogen (H2). Higher VFA productivity was observed at pH 10 operation (6.3 g/l) followed by pH 9, pH 6, pH 5, pH 7, pH 8 and pH 11 (3.5 g/l). High degree of acidification, good system buffering capacity along with co-generation of higher H2 production from food waste was also noticed at alkaline condition. Experiments illustrated the role of initial pH on carboxylic acids synthesis. Alkaline redox conditions assist solubilization of carbohydrates, protein and fats and also suppress the growth of methanogens. Among the carboxylic acids, acetate fraction was higher at alkaline condition than corresponding neutral or acidic operations. Integrated process of VFA production from waste with co-generation of H2 can be considered as a green and sustainable platform for value-addition. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Among the biological routes, acidogenic fermentation (heterotrophic-dark) process for biohydrogen (H2) production shows the promise of practical viability due to its feasibility of utilizing different types of wastes as feedstock. In acidogenic microenvironment, monomers are formed from hydrolysis of organic compounds by

⇑ Corresponding author. Tel./fax: +91 40 27168107. E-mail address: [email protected] (S. Venkata Mohan). http://dx.doi.org/10.1016/j.biortech.2015.01.007 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

hydrolytic microorganisms and thus lead to the formation of H2 along with a mixture of low-molecular-weight organic acids (volatile fatty acids (VFA) or carboxylic acids) and CO2 as major fraction (Venkata Mohan, 2009). Bacterial hydrogen gas (H2) production is the consequence of transfer of cellular reduction equivalents, i.e. electrons (e), onto protons (H+) and hence H2 generation occurs with minimal energy requirement in the anaerobic process (Srikanth and Venkata Mohan, 2014). This H2 when extracted from the system can be used as a fuel but when retained in the system it acts as an electron donor to produce both acetic acid (homoacetogenesis) and methane (hydrogenoclastic methanogenesis)

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(Noori and Saady, 2013) where CO2 is the electron acceptor. Acidogenic bacteria facilitate the formation of acetic acid (C2), propionic acid (C3), butyric acid (C4) and valeric acid (C5), etc., and the higher chain fatty acids (C3 and above) are further oxidized to acetic acid through the action of syntrophic bacteria (H2-producing acetogenic bacteria). Acetic acid can also be produced by homoacetogens utilizing H2 and CO2. H2 and VFA are the two important value added products of acidogenic fermentation which if harvested properly in an integrated approach will make the whole process environmentally sustainable and economically viable. Acidogenic H2 production was well studied and documented with various feedstock and at present it is at the stage of upscaling (Pasupuleti et al., 2014; Chandra and Venkata Mohan, 2014; Venkata Mohan et al., 2008). These short chain carboxylic acids can be utilized further as they are building blocks of various organic compounds including alcohols, aldehydes, ketones, esters, olefins, etc. (Singhania et al., 2013). By implementing appropriate methods, VFA can be also converted to alcohols (Uyar et al., 2009), biohydrogen (Srikanth et al., 2009) bioplastics (Cai et al., 2009; Venkata Mohan et al., 2010; Reddy et al., 2012; Amulya et al., 2014), microalgal lipids (Venkata Mohan and Devi, 2012), bioeletricity (Mohanakrishna et al., 2010), aldehydes (Spirito et al., 2014; Silva et al., 2013), etc. and are also used as preservatives in food and beverage industry and in the synthesis of pharmaceutical/chemicals. Earlier reports documented anaerobic acidification of waste materials viz., urban organic waste, sludge, sugar beet processing waste, vinasse and vegetable waste as primary feed stocks for VFA production with different degrees of success (Singhania et al., 2013; Cai et al., 2004; Dinamarca et al., 2003). However, for the substrate to be viable as feedstock, it should be available in a reasonably good amount and should have high biodegradability and carbon load. Moreover, wastes containing the above conditions if used as a feedstock, shall contribute greatly to the ecological and economic efficiency of the process. In this realm, canteen based food waste which fulfils the above criteria can be thought of as potential feedstock for acidogenic fermentation. One third of the food produced globally for human consumption is wasted and lost accounting to around 1.3 billion tonnes. An attempt was made to study the viability of carboxylic acid synthesis under uncontrolled redox condition through fermentation of food waste coupled with the H2 production towards hydrogen-carboxylate platform development. Open microbiome batch experiments were designed to assess the carboxylic acid synthesis and associated H2 production at variable redox conditions (pH, 5–11). The major objective of this study was to establish optimum redox conditions for higher fatty acid productivity. 2. Experimental methodology 2.1. Anaerobic consortia Anaerobic consortium collected from full scale anaerobic reactor treating sewage wastewater was used as parent inoculum. The sludge (3.6 g VSS/l; 40 ml) after removing grit was enriched with food waste (15 kg COD/m3-day; 48 h; pH, 7) for four cycles prior to inoculating the bioreactors.

arating system that works based on the gravity. Oil interferes in the biological activities of the inoculum and covers the carbohydrate content of the food waste hence making it unavailable for further digestion. The oil free waste was used as a feedstock after adjusting pH and organic load (OL) by diluting with domestic sewage. Chemical oxygen demand (COD) of non-diluted waste has 4900 kg COD/l with a reasonably good biodegradable fraction (BOD/COD) of 0.72. All the experiments were performed at an organic loading rate of 15 kg COD/m3-day. 2.3. Experimental details Seven identical bench scale anaerobic reactors were fabricated using borosilicate-glass bottles to have a total/working volume of 0.5/0.4 l and gas holding capacity of 0.1 l. The reactors were operated in suspended growth configuration in batch mode for 6 cycles. Each batch was operated with 48 h of retention time comprising of 20 min of FILL phase, 47 h of REACT (anaerobic) phase, 20 min of SETTLE phase and 20 min of DECANT phase in sequencing/periodic discontinuous mode. All the reactors were operated at an ambient temperature (28 ± 2 °C) with organic load rate 15 kg COD/m3-day to study the relative efficiency of VFA production as a function of pH. Prior to operation, the pH of each reactor was adjusted to 5, 6, 7, 8, 9, 10, and 11 using 1 N HCl or 1 N NaOH. Nitrogen gas was sparged into the reactor for 5 min after every feeding and sampling event to maintain anaerobic conditions. The reactors were kept in suspension mode during REACT phase by continuous mixing (100 rpm). Prior to startup, all the reactors were inoculated with 10% of inoculum. 2.4. Analysis Carboxylic acid composition was analyzed using high performance liquid chromatography (HPLC; Shimadzu LC10A) employing UV–Vis detector (210 nm) and C18 reverse phase column (250  4.6 mm diameter; 5 lm particle size, flow rate: 0.6 ml/h; wave length: 210 nm). Mobile phase of (40% acetonitrile in 1 mN H2SO4; pH, 2.5–3.0) and 20 ll sample injection was used. Biogas composition was monitored using gas chromatography (GC; NUCON 5765) using thermal conductivity detector (TCD) with 1/ 800 X 2 m Heysep Q column employing Argon as carrier gas. The injector and detector were maintained at 60 °C each and the oven was operated at 40 °C isothermally. Chemical oxygen demand (COD-closed refluxing titrimetric method), VFA and pH were estimated by the standard methods. (APHA, 1998) Buffering capacity (b) was estimated based on the acid-base titrations employing auto-titrator (Mettler Toledo DL50). (Velvizhi and Venkata Mohan, 2014). The sample was divided into two parts of 3 ml each prior to the test. The first part was titrated with 0.1 N HCl till the end point at pH = 1.9 and the second part was titrated against 0.1 N NaOH till the end point of at pH = 12. The buffering capacity (b) was calculated using the equation, where, C is the concentration of acid or base (mol), Vs is the volume of sample (ml), m is the slope of tangent on curve (Eq. (1))

b¼

C Vs  m

ð1Þ

3. Results and discussion 2.2. Waste 3.1. Fatty acids Composite food waste was collected from institute canteen. The collected food waste after removing non-food particles was masticated using electrical blender and then filtered through a stainless steel sieve to remove coarse materials that may cause clogging problems. Oil present in the waste was separated using an oil-sep-

Experiments illustrated variation in total carboxylic acids (VFA) concentration and composition as a function of initial pH with respect to operation time (Fig. 1a). VFA production was analyzed at every 12th h of the operating cycles. It was observed that in

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7000 pH 5

pH 6 pH 9

6000

pH 7 pH 10

pH 8 pH 11

5000 4000 3000 2000 1000 0 0

50

100

150

200

250

300

Time (h) pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11

VFA Production (mg/l)

6000

4500

3000

1500

0 0

10

20

30

40

50

Time (h) Fig. 1. (a) Change in VFA production at different pH variations with respect to time; (b) Change in VFA pattern at different pH studied from 0th h to 48th h during the operation.

all the conditions studied VFA production started without much lag and there was a noticeable increment at 12 h of cycle operation. System operated at initial pH 10 illustrated comparatively higher fatty acids (as VFA; 6.3 g VFA/l) production followed by pH 9 (5.17 g VFA/l), pH 6 (4.5 g VFA/l), pH 5 (4.2 g VFA/l), pH 7 (4.1 g VFA/l), pH 8 (3.8 g VFA/l) and pH 11 (3.5 g VFA/l). By observing the results it can be elucidated that pH plays a major role in fatty acid synthesis. This can be attributed to the fact that most of the acidogens cannot survive in extremely acidic (pH 3) or alkaline (pH 12) environments (Jie et al., 2014; Dinamarca et al., 2003; Singhania et al., 2013). The optimal pH values for the production of VFA fall in the range of 5.25–11, but the specific range depends on the type of substrate used. Further more based on the VFA accumulation and degradation pattern in the results, VFA profiles can be divided into three phases (Fig. 1b). In first phase (12 h) there was a rapid increase in the VFA concentration in all the experimental variations studied, due to the activity of acidogenic bacteria (AB). These AB are fast growing bacteria, with minimum doubling time of around 30 min and are capable of fermenting the soluble organic fraction of substrate within a short span of time. In the second phase (between 12 and 36 h), the rate of production was relatively low in comparison to the first phase. Accumulation of fatty acids alters the system buffering condition leading to retardation of the rate of VFA production. Maintaining a constant pH during the operation is thus expected to retain high VFA production rate for prolonged periods. On the contrary, the third phase was more distinct, it was toward

consumption of fatty acids rather than its accumulation. Consumption/accumulation of fatty acids is generally associated with methanogenesis either by acetoclastic (pH, 6–8) or hydrogenoclastic (pH, 9–10) archea. Acetotrophic methanogenesis utilizes VFA in the form of acetate which undergoes a dismutation reaction to produce CH4 and CO2 (Horiuchi et al., 2002). Hydrogenotrophic methanogenesis utilizes CO2 and H2 for the production of CH4. Fermentation microenvironment particularly in alkaline redox microenvironment favored higher carboxylic acid production compared to corresponding neutral and acidic conditions. In, acidogenic fermentation which comprises four phases (hydrolysis, acidogenesis, acetogenesis and methanogenesis), the first hydrolytic step plays a crucial role as it makes the substrate available to the microbial consortia for the further metabolism (Mohanakrishna and Venkata Mohan, 2013). In this regard the redox condition can have an impact. The food waste generally composed of carbohydrates, proteins and fats, and these complex compounds have to be broken down to simpler molecules to be metabolized (carbohydrates to mono/disaccharides; proteins to amino acids; lipids to fatty acids/glycerol). Compared to acidic pH, an alkaline redox microenvironment enhances the hydrolysis of carbohydrates and proteins by causing the ionization of the charged groups (e.g. carboxylic groups) and therefore, facilitating the availability of soluble and easily assimilable for fermentation, which generally occurs at the initial hour of the fermentation. At high alkaline pH, most of the carbohydrates are anaerobically biodegradable (Noike et al., 1985).

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Propionic acid (mg/l)

Valeric acid (mg/l)

Acetic acid (mg/l)

Butyric acid (mg/l)

a

b Cycle 1 Cycle 4

Cycle 2 Cycle 5

Cycle 3 Cycle 6

VFA Production (mg/l)

6000

5000

4000

3000 0 5

6

7

8

9

10

11

pH Fig. 2. (a) Production profile of individual VFA produced at different pH with respect to different time interval (i) 4 h (ii) 12 h (iii) 24 h (iv) 48 h. (b) Maximum VFA production in different cycle with different pH.

The degradation efficiency of soluble proteins under alkaline and neutral conditions was higher than that under acidic conditions. The alkaline pH is beneficial for the solubilization of proteins and also the degradation of soluble proteins. Protein gets hydrolyzed at alkaline condition due to the availability of OH ion which acts as a nucleophile on the peptide bond resulting in its breakdown and generation of free amino acids. The fats are more soluble if pH value is alkaline (pH 8) compared to the pH of acidifying reactors (5.5–6.0) where the fat is mostly present in insoluble form manifesting low hydrolysis. Also, strong alkaline redox condition prevents the growth of both acetoclastic and hydrogenoclastic methanogens and hence retains fatty acids without converting it to terminal methanogenic end products such as CO2 and CH4. Operation at pH 11 showed not so stable performance due to the prevailing higher alkaline redox microenvironment. Next to alkaline, acidophilic conditions showed good fatty acids production. Low availability of soluble substrate to acidogenic bacteria leads

to less VFA generation at the acidic pH. Neutral pH operation showed relatively lower VFA production due to the dominance of methanogenic activity. Neutral or nearly neutral conditions (6.8– 7.2) are generally optimum for methanogensis (Yan et al., 2010; Venkata Mohan, 2009). Methanogens consume fatty acids along with CO2 and H2 towards CH4 production. Anaerobic metabolism shifts to fatty acids consumption in the pH range 5.0–8.0. 3.1.1. Fatty acid composition Carboxylic acids (short-chain volatile fatty acids; C2 to C5) viz., acetic acid (HAc), butyric acid (HBu), propionic acid (HPr) and isovaleric acid (HVa) were produced (Fig. 2a). Among the carboxylic acids, HAc was detected at a relatively higher fraction (4.2 g/l; pH 10) followed by HBu (1.8 g/l; pH 5), HPr (1.4 g/l; pH 9) and HVa (0.04 g/l; pH 6) (Fig. 2b). System pH and operation time showed marked influence on the rate of production as well as its composition. Acetic acid production was found to be relatively higher in

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alkaline pH operations. Alkaline pH improves the acetic acid accumulation/production and its percentage in the total VFA. With increment in pH from 5 to 10, a consistent increment in production of acetic acid was observed followed by an acute drop at pH 11. Its production was observed until 36 h and thereafter, proceeding with fermentation resulted in its consumption in all the experiment variations studied. However, butyric acid showed a contrary trend. Production and consumption of butyric acid was inconsistent with time and pH. Higher production was observed at acidic pH (5; 1.8 g/l). With rise in pH, a decrement in production rate was noticed until pH 8 which, stabilized until pH 10. However, at pH 11 a sharp rise in its production was noticed. On the contrary, propionic acid showed more or less uniform production at all the redox conditions studied. Consumption was not noticed at pH 6,

7, 8 and 9 operations. Iso-valeric acids synthesis was relatively low compared to other carboxylic acids. However, the trend was quite distinctive, wherein, consumption was observed specifically at 12 h followed by production at 24 h in all the experimental variations studied. At 48 h of operation, valeric acid was not detected indicating its complete consumption in the fermentation process. 3.1.2. Acidification Degree of acidification (DOA) represents the extent of acid formation achieved due to the production of carboxylic acids in relation to substrate (as COD) degradation using the relation (Alkaya and Demirer, 2011; Amulya et al., 2014) (Eq. (2)):

Degree of acidification ðDOA;%Þ ¼ ðSf =Si Þ  100

ð2Þ

a

Biohydrogen (%)

b

50 40

pH 5

pH 6

pH 9

pH 10

pH 7

pH 8

pH 11

30 20 10

Biomethane (%)

0 30

20

10

0 12 h

24 h

48 h

Time (h) Fig. 3. (a) Contribution of individual acid and total degree of acidification in terms of acetic acid, butyric acid and propionic acid at different pH. (b) Biogas composition evolved from the reactor at different pH conditions studied (i) H2 production and (ii) CH4 production.

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where, Si represent initial substrate concentration measured in COD as mg/l and Sf is net VFA concentration (final-initial) expressed as theoretical equivalents of COD concentration (COD equivalents of individual VFA viz., acetic acid, 1.066; propionic acid, 1.512; butyric acid, 1.816) as mg/l. Degree of acidification enumerates the capability of system to produce the carboxylic acids. Degree of acidification was calculated in terms of individual acetic acid, butyric acid and propionic acid concentrations and also with the mixture of three fatty acids (Fig. 3a). As valeric acid was produced in very low concentrations, it was not included in the acidification calculation. VFA composition, fermentation time and initial pH showed a marked influence on the acidification. The degree of acidification was higher with pH 10 operation followed by pH 9, pH 6, pH 5, pH 7, pH 11 and pH 8. It is interesting to note that higher acetic acid concentration contributed to higher acidification (pH 10, 25.29%; pH 9, 21.26%). On the contrary, higher propionic acid and butyric acid concentrations documented relatively lower acidification values (pH 11, 16.81%; pH 7, 18.89%). When acidification was calculated based on individual fatty acids, acetic acid showed highest value at pH 10 (14.45%) followed by pH 9 (10.70%), pH 8 (7.55%), pH 7 (4.74%), pH 5 (3.11), pH 11(2.68%). In the case of butyric acid, pH 5 (10.85%) showed highest acidification values followed by pH 6 (9.71%), pH 7 (7.82%), pH 11(6.76%), pH 9 (5.07%). Conversion of propionic acid was less compared to acetic and butyric acid and highest acidification was recorded at pH 11 (7.38%), followed by pH 5 (6.83%), pH 10 (6.50%), pH 7 (6.33%), pH 9 (5.49%), pH 6 (4.93%) and pH 8 (4.33%). Degree of acidification is largely influenced by the type of the VFA produced in the system. 3.2. Biogas Anaerobic fermentation of wastes generates H2 and CH4 which are two important energy carriers along with CO2. Co-generated biogas showed the presence of H2 and CH4 along with CO2 (Fig. 3b). The composition and quantity of biogas varied with respect to the experimental condition adopted. Highest H2 produced was recorded at 12th h of cycle operation at pH 10 operation (30%) followed by pH 9 (25%), pH 7 (15%), pH 11(10%), pH 6 (9%), pH 5 (4%) and pH 8 (3.5%). Subsequent analysis at 24 h and 48 h showed marked changes. At 24th h, H2 production was found highest at pH 11 operation (21.3%) for one cycle followed by pH 10 (10%) and least for pH 6 (1.6%). At the end of the cycle, traces of H2 were observed at pH 11 operation (11.707%) and least at pH 5 (1%). The H2 production was dependent on the hydrolysis of the organic substrate into simpler compounds. In the absence of external electron acceptors organic compounds get catabolized to energy rich intermediates by substrate level phosphorylation along with hydrolysis. With H2 production a redox balance will be achieved which is one of the means of disposing of excess electrons. Interestingly, higher H2 production was observed at alkaline pH than at acidic/neutral conditions. It should be noted that the H2 production at pH 11.0 was inconsistent even though higher at 24 h, suggesting that strong alkaline pH impaired acidogens. Cai et al. (2004) indicated that the alkaline pH improved the H2 production from solid waste, such as sewage sludge or food waste. Maximum CH4 production was observed at pH 7.0 (21%) followed by pH 8 (13%), pH 9 (4.6%) and pH 10 (2.5%). No traces of CH4 were found at pH 5, 6 and 11 during the initial hour of the operation. A marginal methanogenic activity was observed at the end of the cycle operation for pH 6 and pH 11 operations. Decrement in CH4 was observed in the later hours as the pH approached acidic conditions which inhibited CH4 production to large extent. During acetogenesis VFA are converted to acetate and H2 followed by methanogenesis where acetate, H2 and CO2 are converted to methane and water. It is well known that the optimum pH range for the growth of methanogens is between 6.8 and 7.2, where

CH4 yields are consistent with this range. Methanogenesis removes the semi-final products of anaerobic digestion: H2, small organic compounds, and CO2 without which, a great deal of carbon (in the form of fermentation products) would accumulate in anaerobic environments (Adegunloye et al., 2013; Venkata Mohan, 2009). The VFA consumed for CH4 production only accounts for a very small amount of COD. Otherwise, acidogens will be more active converting organic substrates to VFA and the resulting fatty acids accumulation will then further drop in pH to inhibitory levels. CO2 production was high compared to two other two gases. At 12 h, maximum production was observed at pH 5.0 followed by pH 8.0, pH 6.0, pH 11.0, pH 10.0 and pH 7.0. At 24 h of the cycle operation, the CO2 production was low for above neutral pH. At the end of the cycle CO2 production was found to be similar to 24 h with a slight decrement. Low CO2 concentration in the alkaline pH may be attributed to the presence of the hydrogenotrophic methanogenesis while high due to presence of acetoclastic methanogenesis in below neutral. By forming bicarbonate/carbondioxide buffer system, CO2 positively contributes to system buffering capacity by giving resistance to a pH-change mainly in the digester liquid. 3.3. System redox condition VFA production is influenced by many factors other than substrate degradation making initial pH a critical parameter. The accumulation of short chain fatty acids leads to a pH drop, and their toxicities are higher when the pH is below 7.0 (Hwang et al., 2004). Drop in system pH indicated fatty acid accumulation in the anaerobic system. Change in pH during process operation may influence the enzyme activities and in turn metabolic activities of the biocatalyst. Prior to start-up, pH of the systems were initially adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0. Initial pH greatly influenced the fatty acids synthesis and profile. Decrement in pH was observed towards acidic condition in all the experimental variations studied (Fig. 4a). A rapid drop in pH was noticed until 12th h of operation as the easily digestible fraction of organic matter was hydrolyzed and converted to fatty acids along with CO2 followed by a slight decrease and again increment at the end of cycle period. Increment in system pH at end might be attributed to the degradation of the fatty acids by obligate proton reducing bacteria/methanogens/heterotrophic acetogens. System operating with initial pH 11 was unstable for the initial 3 cycles. Fig. 4b shows the pH change (DpH) as the function experimental conditions and time to enumerate specific redox change in that particular period of time interval. With increase in initial pH from acidic to basic condition, the drop in pH continued up to 24 h of cycle operation, which confirmed the accumulation of fatty acids. However, at 36 h and 48 h, consumption of fatty acids was more evident, indicating shift in pH towards neutral redox condition. 3.3.1. Buffering capacity Fatty acids production also has a significant influence on the system’s buffering capacity (Venkata Mohan et al., 2009). System’s internal buffering capacity was measured for all the pH conditions at different time intervals (Fig. 4c). Buffers in aqueous systems tend to resist changes in pH when small amounts of acid (H+) or base (OH) is added. Initially buffering capacity in all the reactors increased until 24th h and decreased later except for the pH 10 operation. At 12th h, maximum BC (0.222 bmol) was observed at pH 8 while for other pH conditions it was in the range of 0.0058 to 0.0183 bmol. At 24th h, highest BC was found at pH 10 (0.030 bmol) followed by pH 11 (0.029 bmol), pH 6 (0.024 bmol), pH 9 (0.022 bmol), pH 8 (0.021 bmol), pH 5 (0.018 bmol) and pH 7 (0.014 bmol). System’s BC correlated well with the total VFA production. More the fatty acids production, higher the system BC,

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which was specifically observed with pH 10 operations. CO2 is also liberated along with fatty acid and H2 during fermentation by acidogenic as well as acetogenic microbes. The system showed improved buffering until 24th h of cycle operation, which can be attributed to CO2 production which helps in system buffering through carbonate formation. Accumulation and/or consumption of fatty acids directly influence the system BC. By the end of the cycle period, highest BC was observed at pH 10 operation (0.025 bmol) followed by pH 11 (b 0.015), pH 7 (b 0.012), pH 5 (b 0.010), pH 6 (b 0.0096), pH 9 (b 0.009) and pH 8 (b 0.0074). Consumption of accumulated VFA by the homoacetogens and metha-

a

12

pH 5

pH 6

nogens resulted in improved BC at the later hours (pH 6, 0.0096 bmol; pH 9, 0.009 bmol; pH 8, 0.0074 bmol). With initial pH 7 and pH 10 operation, buffering increased with time and drop was observed at the end of the cycle. The buffering capacity of the biological systems is also associated with in situ produced volatile buffers in the anaerobic system. The pH is kept stable by the buffer effect of the protein residues and other macromolecules (Dinamarca et al., 2003). Fermentation of proteins leads to production of ammonia which facilitates formation of ammonium bicarbonate (pKa 6.35/9.35) in the presence of CO2, which supplements additional in situ buffer to the operating system. At

pH 7

pH 8

pH 9

pH 10

pH 11

11 10 9

pH 8 7 6 5 4 0

50

100

150

200

250

300

9

10

11

Time (h)

b

pH 1 5

6

7

8

0

ΔpH

-1

-2

-3

-4

Δ pH 12

(12-0)

Δ p H 24 (24-0)

Δ pH 36

(24-36)

Δ p H 48 (36-48)

-5

β

c

Fig. 4. (a) Change in pH pattern during the fermentation of food waste of individual reactor when operated at uncontrolled pH. (b) Influence of initial pH on DpH calculated at different time interval to compare the change in pH at different pH studied. (c) Buffering capacity of the system operated at different pH (calculated based on the slope of tangent on curve at different time interval).

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alkaline pH conditions, free ammonia present in the system, which in association with bicarbonate (pKa 6.35/9.35), carbonate (pKa 6.35/9.25) and fatty acids viz., formic acid (pKa 9.25) and acetic acid (pKa 4.75/9.25) plays a great role in buffering these biological systems. The proper balance between the ammonia and carbonate/ bicarbonates/fatty acid leads to enhanced buffering of the system. Low concentration of acids at acidic pH range attributes to low BC compared to the alkaline conditions. 3.4. Yields and conversion efficiency Carboxylic acid production and consumption pattern as a function of fermentation time helps to understand the dynamics of acidogenic process occurring within the system. The interactions between the acidogenic bacteria, acetogens, homoacetogens and methanogens manifest VFA accumulation and/or consumption. The production/consumption rates of carboxylic acids are calculated based on the following equations proposed (Eqs. (3) and (4)).

Production rate of carboxylic acids ðPRCa Þ ¼ ðVFAmax  VFAint Þ=T Prod

ð3Þ

Consumption rate of carboxylic acids ðCRCa Þ ¼ ðVFADrop  VFAmax Þ=T Drop

ð4Þ

where, VFAmax represents maximum VFA concentration (g/l), VFAint is initial VFA concentration (g/l), VFADrop denotes drop/consumption in VFA concentration due to its consumption (g/l), TProd is production time in hours and TDrop represents concentration dropping/ consumption time (hours). Table 1 depicts the production and consumption rates of individual carboxylic acids as a function of fermentation time. Production was calculated from the initial hour to the time of highest fatty acids accumulation while the consumption was calculated from the point of degradation to end of the cycle. The positive and negative values explain the rate of production of carboxylic acids and consumption respectively (Table 1). Analysis of the data showed some interesting observations. Increasing trend in PRCa was observed with HAc from pH 5 to pH 10 operations, which dropped in pH 11 operation. Rate of HAc production was lower at acidic and neutral pH compared to alkaline redox condition (pH 10 and pH 9 operations). Highest production rate was registered at pH 10 operation (+0.121 g/h). Higher production rate with HBu was observed between pH 5 and 7 operations, indicating feasible condition for its synthesis. In the case of HPr, the production rate was inconsistent in the pH spectrum studied. In the overall study HVa had least PRCa at all pH conditions studied. CRCa had distinct variations with all the carboxylic acids observed during fermentation. CRCa with HAc was found to be moderate in operations with initial pH 5 (0.026 g/h), pH 6 (0.025 g/h), pH 8 (0.021 g/ h), pH 10 (0.033 g/h) and pH 11 (0.022 g/h) when compared to those with pH 7 (0.060 g/h) and pH 9 (0.086 g/h). CRCa of HPr was negligible at pH 6, pH 7 and pH 8 operations which influenced

the total fatty acid production and degree of acidification. Higher CRCa with HPr was observed at pH 5 (0.038 g/h), pH 9 (0.038 g/ h), pH 10 (0.014 g/h), and pH 11 (0.037 g/h) operations. CRCa of HVa was observed in all the operating systems. Comparing the pH spectrum studied, the consumption of HAc was high followed by valeric acid. On the contrary, HPr and HBu showed production only at pH 6, 7 and 8 and pH 7 operations, respectively illustrating that these conditions are not so feasible for their consumption (Fig. 5). At acidic initial pH operations, pH favored the production of longer chain fatty acids, since more reducing equivalents are available that can be incorporated into the fatty acid chains (Zoetemeyer et al., 1982). Also at low pH, fatty acids in their undissociated form have the ability to diffuse freely across lipid bilayers and liberate protons in the cytoplasm lowering the cytoplasmic pH (Booth, 1985). At low cytoplasmic pH anions accumulate inside the cell (Russell and Diez-Gonzalez, 1998) and undissociated acid intercalates within the lipid bilayer (Stratford and Anslow, 1998) disturbing the metabolic activities of the microbes. At external acidic pH, the acetic acid that is prevalent (pKa = 4.76) in its nonionic form passes through the membrane resulting in an overload of acetic acid inside the cell. Accumulation of acetate inside in the cell causes inhibition of the phosphoroclastic pathway leading to the production of butyric acid. Initially two acetyl CoA molecules condense to form acetoacetyl CoA which in with few further steps reduce to butyric acid, leading to high butyric acid concentration at acidic pH. This phenomenon was evident in this study particularly below neutral pH operation. The pH strongly influences the relative amount of VFA. pH range between 4.0 and 5.0 is favorable for propionate production (H2 sink reaction) while at pH 6.0–7.0, acetate and butyrate are major formation favored at a transition zone between pH 5.0 and 6.0. On the contrary, at alkaline conditions (pH > 7) fatty acids in ionic form are not able to pass through lipid bilayer which leads to their accumulation outside the cell (Lier et al., 1993). The phosphoroclastic pathway dominates at alkaline conditions resulting in increased acetate as well as H2 production. The experimental data correlated with the acetate production (comparatively higher) observed at pH 10.0. Propionate and butyrate get degraded thermodynamically only when acetate and especially H2 are effectively eliminated by the MB (Stams et al., 1992). Propionate gets catabolized anaerobically to acetate and CO2. However, catabolism may get inhibited in the presence of other VFAs and the extent of inhibition depends on the fatty acid concentration and the system pH (Siegert and Banks, 2005). The catabolism of propionate is endergonic in nature, while methanogenic conversion of acetate is exergonic. In general methanogenic oxidation of acetate stimulates propionate degradation thermodynamically but when concentrations of butyrate and acetate are increased, removal of propionate decreases. Acetogenic bacteria keeps symbiotic relation and take profit from both hydrogenotrophic methanogens (reducing H2 partial pressure) and acetoclastic methanogens. Acetate removal has an influence on the energetics of VFA oxidizing reactions, espe-

Table 1 VFA Production (0–36th h) and consumption rate (36–48th h) during acidogenic fermentation of food waste. Initial pH

5 6 7 8 9 10 11

PRCa (g/h)

CRCa (g/h)

HAc

HPr

HBu

HVa

HAc

HPr

HBu

HVa

+0.021 +0.047 +0.036 +0.061 +0.088 +0.121 +0.018

+0.040 +0.028 +0.036 +0.025 +0.031 +0.038 +0.043

+0.055 +0.049 +0.039 +0.017 +0.024 +0.021 +0.033

+0.0001 +0.0005 +0.0004 +0.00003 +0.00009 +0.0003 +0.00006

0.026 0.025 0.060 0.021 0.086 0.033 0.022

0.038 +0.042 +0.023 +0.003 0.038 0.014 0.037

0.073 0.039 +0.034 0.001 0.004 0.009 0.046

0.0016 0.0028 0.0024 0.0016 0.0019 0.0022 0.0015

‘+’ represents production of fatty acids; ‘’ represents consumption of fatty acids.

VFA (max, g/l)

Acid

4.2 4.5 4.1 3.8 5.1 6.3 3.5

20.79 20.84 18.89 15.72 21.26 25.29 16.81

Deg

(%)

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S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113

a

0.1 Production rate Consumption rate

0.1

0.05

0

5

6

7

8

9

10

11

-0.05

Butyric acid production/consumption rate (g/h)

Acetic acid production/consumption rate (g/h)

0.15

0.05

0 5

7

8

9

10

11

-0.05

-0.1

-0.15

-0.1

pH

pH

0.0009

0.06

0.03

0

5

6

7

8

-0.03

-0.06 pH

9

10

11

Valeric acid production/consumption rate (g/h)

0.09

Propioic acidproduction/consumption rate (g/l)

6

0

5

6

7

8

9

10

11

-0.0009

-0.0018

-0.0027

-0.0036

pH

b

Fig. 5. (a) Profiles showing the production and consumption rate of individual fatty acids with the function of pH and time operation. (b) Role of microorganisms at variable redox microenvironment for acidogenic fermentation of food waste to VFA along with co-generation of biohydrogen: an overview.

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cially during iso-valerate degradation, where three molecules of acetate and only one molecule of H2 are formed. This might explain why iso-valerate degradation was found in all the systems. Higher H2 production was observed at 24th h of the cycle after which, it decreased at alkaline pH operation. When H2 concentration was high, proper proton and electron flow is essential where MB play an important and regulatory role. H2 gets consumed by hydrogenotrophic methanogenesis as low H2 and CO2 concentration which was specifically observed with pH 9 and 10 operations at the later hours. Hydrogenotrophic methanogenesis creates an electrochemical gradient across cell membrane, which, is used to generate ATP through chemiosmosis (Goyal et al., 2014). The inhibition of hydrogenotrophic methanogenesis leads to H2 accumulation which in turn inhibits acetogenesis (Zaher et al., 2004). The consumption of H2 by MB provides a low partial H2 pressure, which enables the obligate proton reducing bacteria to degrade the organic acids, which are thermodynamically very unfavorable reactions and hence at alkaline pH along with hydrogenotrophic methanogesis, acid consumption was also observed. Along with this homoacetogenesis may also produce acetate, which consumes H2 and CO2 counter balancing the acetate accumulation and degradation. The conversion of H2 and CO2 into acetate is energetically more favorable at higher H2 partial pressures. At pH 7, higher CO2 and CH4 composition leads to the presence of acetoclastic methanogenesis, which utilizes fatty acids in the form of acetate which undergoes a dismutation (simultaneous oxidation and reduction) reaction to produce CH4 and CO2. Acetotrophic methanogenesis is enzymatically catalyzed electron transfer from the carbonyl group (e donor) of the carboxylic group to the methyl group (e acceptor) of acetic acid to produce CO2 and CH4 gas. At acidic pH conditions presence of high CO2 and traces of H2 are attributed to the presence of hydrolytic activity of the acetogens, acidogens and the basic metabolic products of the mixed consortium.

3.5. Waste remediation Substrate degradation (based on the COD removal) showed distinct variation in the treatment efficiencies as a function of initial pH (Sfig. 1). Substrate degradation efficiency was higher at neutral pH operation (pH 7, 66%) followed by pH 8 (62%), pH 9 (61%), pH 10 (55%), pH 6 (51%), pH 5 (44%) and pH 11 (30%) operations. Due to the prevalence of acetoclastic methanogenic activity, highest substrate degradation was observed at pH 7 and 8 operations. At pH 10 operations, due to presence of the hydrogenoclastic methanogenic activity there was less removal and more accumulation of acids in the form of COD. Less methanogenic activity and less availability of substrate may be the reason for less COD removal observed at pH 5, 6 and 11 operations.

4. Conclusions Experiments documented the possibility of VFA production at alkaline pH condition associated with co-generation of H2 during fermentation of foodwaste. Data illustrated the role of initial pH on both carboxylic and H2 production. At different initial pH operations, the interactions between the acetogens, homoacetogens and methanogens manifest VFA accumulation and/or consumption. To enhance acidification further, the consequence of the drop in pH at the later hours of operation need to be addressed. Acidogenic fermentation of waste towards hydrogen-carboxylic platform documents a new dimension for economical production of value added products from waste in the framework of biorefinery.

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