Biohydrogen production from co-digestion of cow manure and waste milk under thermophilic temperature

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    Co-digestion

    omn, we ten pVS

    (P < 0.05). Inclusion of waste milk enhances hydrogen production from cow manure and could offeradded benet of waste milk treatment and disposal.

    2012 Elsevier Ltd. All rights reserved.

    ing coe envireplacgen is o

    and organic acid from organic substrates instead of methane (Chenet al., 2002; Perera and Nirmalakhandan, 2010). Model substratessuch as glucose and sucrose are preferred choices for dark fermen-tation; however they are not economically feasible for large scaleproduction. Carbohydrate-rich wastes have been shown to be suit-able substrates (Lay et al., 1999; Valdez-Vazquez et al., 2005).

    (Diehl and Lapara, 2010). It is plausible; therefore, that milk fromcow treated with antibiotics for mastitis will contain substantialquantities of antibiotic resistant bacteria. The milk is not normallyused for about one week and often discarded to the environment.Such practice does not only promote the spread of antibiotic resis-tant bacteria present in the milk, but also encourage horizontaltransfer of resistant genes to indigenous bacteria within the vicin-ity of the farm (Alonso et al., 2001). This may present a threat topublic health. Anaerobic digestion could be used as a potential toolto reduce the number of resistant bacteria being introduced to the

    Corresponding author. Tel.: +81 155 49 5515; fax: +81 155 49 5519

    Bioresource Technology 110 (2012) 251257

    Contents lists available at

    T

    elsE-mail address: umetsu@obihiro.ac.jp (K. Umetsu).tives to fossil fuels. Its use in fuel cell produces no CO2, a primaryculprit in climate change.

    Dark fermentation is one of the sustainable methods of hydro-gen production. Hydrogen is produced as by-product with organicacid during acidogenic phase of anaerobic digestion process. Theproduced hydrogen is readily consumed by methanogenic hydro-gen consumers to form methane, the nal product of anaerobicdigestion process. Dark fermentation, therefore, involves elimina-tion of hydrogen-consuming methane formers from anaerobicdigestion system through appropriate pretreatment, such as acid,alkaline and heat, of feed and produces hydrogen, carbon dioxide

    potential use of co-digestion of animal manure and carbohy-drate-rich feed to produce hydrogen has been previously suggested(Zhu et al., 2009). There are some reports on co-digestion of animalmanure and other wastes/feedstock for hydrogen production(Perera and Nirmalakhandan, 2010; Yokoyama et al., 2010;Gilroyed et al., 2010). However, there has not been any reportedwork on biohydrogen production from co-digestion of cow manureand waste milk (mastitic milk from antibiotic-treated cow).

    Mastitis, a common and costly disease of dairy cattle, is oftentreated with therapeutic use of antibiotics. Whenever antibioticsare used, antibiotic resistant bacteria are selected and/or evolvedCow manureWaste milkAntibiotic resistant bacteria

    1. Introduction

    Depletion of fossil fuels and growimpact of their increased use on thin call for their partial or completenon-polluting energy sources. Hydro0960-8524/$ - see front matter 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.102ncerns about negativeronment have resultedement with renewable,ne of the ideal alterna-

    Effective hydrogen production often requires supplementation ofan adequate amount of pH buffer and mineral, which will inevita-bly increase the cost of production (Zhu et al., 2008). Co-digestionof several wastes with complementary characteristics could pro-vide balanced nutrient and the required buffering capacity, therebyreducing the cost for pH control or nutritional supplements. TheKeywords:Biohydrogen production

    mulated in M:WM 50:50, 30:70 and 10:90. Overall reduction of more than 90% of cefazolin resistant bac-teria was observed in all the treatments. The reduction was higher at 40 and 60 than 20 g VS L1Biohydrogen production from co-digestiounder thermophilic temperature

    Suraju A. Lateef, Nilmini Beneragama, Takaki YamasKazutaka Umetsu Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Vet

    a r t i c l e i n f o

    Article history:Received 24 November 2011Received in revised form 16 January 2012Accepted 18 January 2012Available online 28 January 2012

    a b s t r a c t

    Biohydrogen production frcows treated with cefazolivolatile solid (VS) L1 wer10:90 and 0:100. Hydroge59.5 mL g1 VS fed at 40 g

    Bioresource

    journal homepage: www.ll rights reserved.of cow manure and waste milk

    o, Masahiro Iwasaki, Chun Ying,

    ry Medicine, Obihiro, Hokkaido 080-8555, Japan

    co-digestion of cow manure (M) and waste milk (WM), milk from mastitisas evaluated in a 3 5 factorial design. Organic loading of 20, 40 and 60 gsted at temperature of 55 C using M:WM (VS/VS) 70:30, 50:50, 30:70,roduction increased with organic loading and M:WM to a maximum ofL1 in M:WM 70:30. Butyrate was the main volatile fatty acid (VFA) accu-

    SciVerse ScienceDirect

    echnology

    evier .com/locate /bior tech

  • charged to the pit, is obtained from a herd of lactating Holsteincows and collected daily from concrete oor of free stall barn. To

    20, 40 and 60 g VS L1 (OL20, OL40 and OL60). The 15 treatmenttypes were tested in triplicate using 1-L lab-scale batch digesters.

    After incubation, the formed colonies were counted and calculatedas colony forming unit per gram of dry matter (cfu g1.DM).

    ical procedure was described in detail by Kimura et al. (1994).Statistical analyses were performed using SAS version 9.2.

    TechThree groups of experiments were conducted. Each group con-sisted of the ve mixing ratios replicated three times and one or-ganic loading. Digesters were manually agitated twice a day andmaintained at 55 C in a water bath for total of ve days for eachgroup of experiment.

    For each treatment, heat-treated manure was combined withwaste milk and mixed with distilled water (Wako Pure Chemi-cal Industries Limited, Japan) to produce 2 L of slurry with thedesired manure:waste milk ratio. The slurry was thoroughlymixed with hand mixer. 600 mL of slurry was added to each di-gester for each replicate. The digesters were ushed with argongas prior to sealing. Gas bag was xed to each digester to col-lected biogas evolved. The digesters were placed in a waterbath. Slurry samples were taken before and after experimenta-remove straws, manure was sieved with 1.6 mm sieve. Prior tobatch runs of experiments, manure was heat-treated as describedby Gilroyed et al. (2010) in order to inactivate methanogens. Sub-sequently, it was stored at 4 C until use (

  • M5

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    0 24 48 72 96 120Time (hr)

    S.A. Lateef et al. / BioresourceHydrogen yields per total VS fed to the digesters are shown inFig. 2. Hydrogen production based on total VS fed to the digestershowed a signicant 2-factor interaction between mixing ratioand organic loading (P < 0.001). The production increased linearly(P < 0.001) with increasing organic loading, and was greater at highorganic loading (OL40 and OL60) than low organic loading(P < 0.05) for all M:WM ratios except M:WM 70:30 and 0:100(P > 0.05). Similarly, at OL40 and OL60, increasing waste milk con-centration signicantly increased production (P < 0.05), except forM:WM 0:100 and treatment with inhibition (M:WM 10:90 atOL40). However, compared to M:WM 70:30, the production forM:WM 50:50, 30:70 and 10:90 at OL20 were not signicantly high-er (P > 0.05). The greatest production (59.5 mL g1 VS fed) was ob-tained at OL40 for M:SW 30:70.

    Hydrogen yield observed in this study appeared to be a functionof both substrate concentration and availability of suitable sub-strates. The results reveal two important points. Firstly, increasing

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    Fig. 1. Hydrogen concentrations in the gas produced at organic loading of 20 g VS L1 (

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    OL20

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    Fig. 2. Hydrogen yield based on total VS fed to the digesters. OL20-organic loadingof 20 g VS L1, OL40-organic loading of 40 g VS L1 and OL40-organic loading of 40 gVS L1. Values are means with standard error.the organic loading increased the hydrogen yield. This marked in-crease was expected, since high substrate concentrations poten-tially lead to high productions (Hallenbeck, 2009). The yield

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    :WM0:50

    M:WM30:70

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    a), 40 g VS L1 (b) and 60 g VS L1 (d). Values are means with standard error bars.

    nology 110 (2012) 251257 253increased with increasing organic loading up to 40 g VS L . Therewere evidences of reduction in hydrogen yield as organic loadingwas increased above 40 g VS L1 (Fig. 2). Accumulation of hydrogenand VFA can cause inhibition of anaerobic degradation process(Argun et al., 2008). The observed reduction was probably causedby higher concentration of total VFA at organic loading of 60 gVS L1 as compared to 40 g VS L1 treatment. The results indicatethat optimal organic loading from this study was 40 g VS L1. Thisoptimal organic loading is in disagreement with previous study(Gilroyed et al., 2010), in which optimal organic loading for hydro-gen production from co-digestion of cattle manure and specic riskmaterial was found to be 20 g VS L1. The difference between val-ues obtained from these studies might be due to characteristics offeedstock used and microbial communities presented in eachstudy.

    The results also show that increasing the concentration wastemilk in the mixture was benecial for hydrogen production. How-ever, the effect was only signicant at organic loading of 40 and60 g VS L1. At these organic loadings, hydrogen yield signicantlyincreased as the quantity of waste milk increased, indicating thatwaste milk has higher hydrogen production potential than manure.However, insignicant yield was achieved with digestion of wastemilk alone (Fig. 2). Preliminary experiments on hydrogen produc-tion from cow manure alone also showed that the productionwas low (data not shown). These highlight the importance of co-digestion of the two substrates. The observed enhancement inyield could be due to positive synergism established in the mix-tures and the supply of balanced nutrients by two substrates, asalso observed by Perera and Nirmalakhandan (2010). They testedthe hypothesis that fermentative hydrogen production from organ-ic-rich feedstock could be enhanced by supplementing with cattlemanure and reported that improved hydrogen productionobserved when cattle manure was co-digested with sucrose was

  • largely due to indigenous hydrogen-producing organisms, bal-anced nutrition and buffer supplied by cattle manure. The opti-mum cow manure to waste milk ratio based on hydrogen yieldwas 30:70 (VS/VS). Maximum hydrogen yield (59.5 mL g1 VSfed) obtained at this ratio is higher than values reported by priorstudies of hydrogen production from batch co-digestion of differ-ent substrates at thermophilic temperature. Lower value(37.5 mL g1 total VS added) was obtained when cassava stillagewas co-digested with excess sludge (Wang et al., 2011) whileGilroyed et al., (2010) reported a maximum yield of 33.1 mL g1 to-tal VS added from co-digestion of cattle manure and specied riskmaterials.

    3.2. Methane concentration

    M:WM M:WM M:WM

    254 S.A. Lateef et al. / Bioresource Tech0

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    70:30 50:50 30:70M:WM10:90

    M:WM0:100Methane concentrations at OL20 for all M:WM ratios are shownin Fig. 3. Traces of methane gas (concentration: 0.22%) were alsoobserved at OL40 for M:WM 70:30, 50:50 and 30:70 while nomethane gas was found at OL60 for all M:WM ratios (data notshown). At OL20 (Fig. 3), the concentration was 7.9% for M:WM70:30 treatment at 24 h and subsequently increased to 21.7 and22.4% at 48 and 72 h, respectively. Similarly, the concentrationfor M:WM 50:50 increased from 9.2% at 24 h to 12.1% at 48 h.However, it decreased in subsequent times until it reached 2.3%at 128 h. Similar trend was observed in M:WM 30:70 and 10:90treatments. Generally, at OL20, increasing the concentration ofwaste milk as a substrate reduced the concentration of methanein the gas produced. Similarly, at OL20, more acidic nal pHs wereobserved as concentration of waste milk increased (Table 1). Thesetwo observations, thus, suggest that increasing the concentrationof waste milk stimulated fast generation of VFA which probably re-duced pH to critical levels for methanogens.

    Inhibition of methanogenesis is essential for effective hydrogenproduction from anaerobic digestion using mixed cultures. Variousinitial pretreatments of seed, such as heat shock, acid, base, aera-tion, freezing and thawing, to inhibit methanogens have been em-ployed (Wang and Wan, 2008). Heat shock pretreatment (HSP) iswidely used for short period operations, as also in this study. De-spite HSP of manure, measurable methane was observed at organicloading of 20 g VS L1. This indicates that effective inactivation ofmethanogens in the microora could be due not only to the heatpretreatment, but also other critical factors, particularly initialpH of the substrates. It has been reported that both heat treatmentand the lower pH (6.2) were required to maximize biologicalhydrogen production in batch tests (Oh et al., 2003). In addition,overloading batch reactors with organic matter would result inlarge VFA concentration which reduces pH to critical levels forFig. 3. Methane concentrations in the gas produced at organic loading of 20 gVS L1. Value are means with standard error bars.methanogens (Valdez-Vazquez and Poggi-Varaldo, 2009). The ini-tial pH values of the substrates were not adjusted. High concentra-tion of VFA likely account for lower pH values observed withorganic loading of 40 and 60 g VS L1 as compared to 20 g VS L1

    (Table 1). The observed methane production at 20 g VS L1 was aresult of high pH values of the treatments. Liu et al. (2006) simi-larly reported that high methane production was observed at ini-tial pH values of 77.5 despite HSP of the inoculum duringhydrogen production from household solid waste in batch test. Re-duced hydrogen production observed at this organic loading couldbe attributed unsuccessful inhibition of methanogenesis. The re-sults of this study thus suggest that manure to waste milk ratioand organic loading (above 20 g VS L1) could be used to adjust ini-tial pH to critical levels for methanogen and hydrogen productionin batch test.

    3.3. VS degradation and VFA production

    Percentage of VS degraded and characteristics of manure:wastemilk substrates are presented in Table 1. There was a signicant(P < 0.001) mixing ratio by organic loading interaction for percent-age of VS degraded. Percentage of VS degraded varied linearly(P < 0.05) and quadratically (P < 0.05) with increased portion ofwaste milk in the mixtures. Similarly, it showed both linea...

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