Bioresource Technology 110 (2012) 251–257

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Bioresource Technology

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

Suraju A. Lateef, Nilmini Beneragama, Takaki Yamashiro, Masahiro Iwasaki, Chun Ying,Kazutaka Umetsu ⇑Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

a r t i c l e i n f o a b s t r a c t

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

Keywords:Biohydrogen productionCo-digestionCow manureWaste milkAntibiotic resistant bacteria

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.102

⇑ Corresponding author. Tel.: +81 155 49 5515; faxE-mail address: umetsu@obihiro.ac.jp (K. Umetsu)

Biohydrogen production from co-digestion of cow manure (M) and waste milk (WM), milk from mastitiscows treated with cefazolin, was evaluated in a 3 � 5 factorial design. Organic loading of 20, 40 and 60 gvolatile solid (VS) L�1 were tested at temperature of 55 �C using M:WM (VS/VS) 70:30, 50:50, 30:70,10:90 and 0:100. Hydrogen production increased with organic loading and M:WM to a maximum of59.5 mL g�1 VS fed at 40 g VS L�1 in M:WM 70:30. Butyrate was the main volatile fatty acid (VFA) accu-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 L�1

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

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Depletion of fossil fuels and growing concerns about negativeimpact of their increased use on the environment have resultedin call for their partial or complete replacement with renewable,non-polluting energy sources. Hydrogen is one of the ideal alterna-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 final 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 dioxideand 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).

ll rights reserved.

: +81 155 49 5519.

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. Thepotential 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 evolved(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

252 S.A. Lateef et al. / Bioresource Technology 110 (2012) 251–257

environment. It has been used to treat biowastes that containpathogenic and antibiotic resistant bacteria (Kunte et al., 1998;Ghosh et al., 2009). However, its use under hydrogen-producingconditions for treating biowastes has not been well studied.

In the present study, co-digestion of cow manure and wastemilk was investigated in batch experiments to examine the effectsof various cow manure to waste milk ratios and different organicloading on; (i) hydrogen production potential and (ii) reductionof cefazolin resistant bacteria. The information from the experi-ments would be beneficial to determine the optimum amount ofwaste milk to be co-digested with cow manure and appropriateloading rate for effective hydrogen production, as well as potentialuse of hydrogen-producing conditions for treatment of cefazolinresistant bacteria.

2. Methods

2.1. Materials

Cow manure was obtained from reception pit of biogas plant atObihiro University, Obihiro, Hokkaido, Japan. Cow manure, dis-charged to the pit, is obtained from a herd of lactating Holsteincows and collected daily from concrete floor of free stall barn. Toremove 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 (<48 h) and evaluated forVS content according to Standard Methods (Standard Methods,2005).

In Obihiro University, cefazolin, a b-lactam antibiotic, whichsuppresses the growth of bacteria by inhibiting cell wall synthesis(Kotra and Mobashery, 1998), is frequently used to treat the cowswith mastitis. Milk from cows treated for mastitis is usually notused for one week and discarded as waste (Galal Abdel Hameedet al., 2006). Waste milk was obtained on the second day afterthe cows were given cefazolin antibiotic. The milk was immedi-ately stored at 4 �C for 7 days before use.

2.2. Experimental design and procedure

A 3 x 5 factorial design consisting of two variables (mixing ratioand organic loading) was used for the experiments. The five ratiosof manure:waste milk (M:WM) tested were 70:30, 50:50, 30:70,10:90, 0:100 (VS/VS) while the three organic loading tested were20, 40 and 60 g VS L�1 (OL20, OL40 and OL60). The 15 treatmenttypes were tested in triplicate using 1-L lab-scale batch digesters.Three groups of experiments were conducted. Each group con-sisted of the five 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 five 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 flushed with argongas prior to sealing. Gas bag was fixed to each digester to col-lected biogas evolved. The digesters were placed in a waterbath. Slurry samples were taken before and after experimenta-tion and analyzed for pH, VS degradation, volatile fatty acid(VFA) and population densities of total and cefazolin resistantbacteria.

2.3. Culturing bacteria

Mixture samples were taken before and after digestion. Sampleswere immediately kept at 4 �C and the analysis was done in lessthan 24 h. Total and cefazolin resistant bacteria were determinedby plate counts on agar media with and without cefazolin at therate of 50 mg L�1. To culture bacteria, peptone, tryptone, yeastand glucose (PTYG) agar (a non-selective medium) was preparedfrom 0.25 g of peptone, 0.25 g of tryptone, 0.5 g of yeast extract,0.5 g of glucose, 0.03 g of MgSO4.7H2O, 0.0035 g of CaCl2.2H2Oand 15 g of Bacto agar per liter to which cycloheximide(100 mg L�1) was added as a fungicide (Kobashi et al., 2005). Dilu-tion plate method was used to determine population densities oftotal and cefazolin resistant bacteria of the samples. Samples werediluted by 10-fold dilutions with phosphate buffered saline (pH7.4). The dilution plate method was conducted in three replicatesand aliquot of 100 lL of sample was spread on the surfaces of threeagar plates. The cultured plates were incubated at 30 �C for 7 days.After incubation, the formed colonies were counted and calculatedas colony forming unit per gram of dry matter (cfu g�1.DM).

2.4. Analyses

Total gas production was monitored daily. Volume of gas pro-duced was measured by wet gas meter. All gas measurements wereexpressed at 0 �C and a pressure of one atmosphere. Prior to mea-suring the volume of gas in the gas bag, gas sample (1 mL) was col-lected from the gas bag using a gas tight micro syringe andanalyzed for gas composition with gas chromatograph (GC)(Shimadzu GC-14A) equipped with a thermal conductivity detector(stainless column and Porapak Q packing). The operational temper-atures of injector port, column and the detector were 220, 150 and220 �C, respectively. Argon was the carrier gas at a flow rate of50 mL min�1.

Volatile solids (VS) were measured according to the StandardMethods (part 2540G Standard Methods, 2005). The pH was mea-sured with a Horiba D-55 pH-meter. Slurry samples were analyzedfor volatile fatty acids [VFA] (acetic, propionic, butyric acid and for-mic acids) with a high performance liquid chromatograph (HPLC,Shimadzu LC-10AD) and Shim-Pack SCR-102H column. The analyt-ical procedure was described in detail by Kimura et al. (1994).

Statistical analyses were performed using SAS version 9.2.

3. Results and discussion

3.1. Hydrogen concentration and production

Time courses of hydrogen concentrations in produced gas areshown in Fig. 1. Hydrogen concentrations over time increased withincreasing organic loading and inclusion of waste milk as substrate.The greatest concentration was 48.7% which was achieved at OL60with M:WM 10:90. This was closely followed by 48.5% from M:SW30:70 also at OL60. This Maximum concentrations in all treatmentswere observed after second day except for M:WM 70:30. No hydro-gen production was observed for M:WM 0:100 except at OL60,where trace of hydrogen gas was observed.

The observed increase in hydrogen concentration in producedgas was expected, since increasing substrate concentration couldincrease the ability of hydrogen-producing bacteria to producehydrogen during fermentative hydrogen production (Van Ginkelet al., 2001). Similarly, the increase might be attributed to the char-acteristics of waste milk. Waste milk contains large fraction of eas-ily degradable VS, which probably accounted for increasedconcentrations of hydrogen gas in the produced gas.

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Fig. 1. Hydrogen concentrations in the gas produced at organic loading of 20 g VS L�1 (a), 40 g VS L�1 (b) and 60 g VS L�1 (d). Values are means with standard error bars.

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Fig. 2. Hydrogen yield based on total VS fed to the digesters. OL20-organic loadingof 20 g VS L�1, OL40-organic loading of 40 g VS L�1 and OL40-organic loading of 40 gVS L�1. Values are means with standard error.

S.A. Lateef et al. / Bioresource Technology 110 (2012) 251–257 253

Hydrogen yields per total VS fed to the digesters are shown inFig. 2. Hydrogen production based on total VS fed to the digestershowed a significant 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 significantly 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 significantly high-er (P > 0.05). The greatest production (59.5 mL g�1 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

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 yieldincreased with increasing organic loading up to 40 g VS L�1. Therewere evidences of reduction in hydrogen yield as organic loadingwas increased above 40 g VS L�1 (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 L�1 as compared to 40 g VS L�1 treatment. The results indicatethat optimal organic loading from this study was 40 g VS L�1. 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 specific riskmaterial was found to be 20 g VS L�1. 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 beneficial for hydrogen production. How-ever, the effect was only significant at organic loading of 40 and60 g VS L�1. At these organic loadings, hydrogen yield significantlyincreased as the quantity of waste milk increased, indicating thatwaste milk has higher hydrogen production potential than manure.However, insignificant 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

254 S.A. Lateef et al. / Bioresource Technology 110 (2012) 251–257

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 g�1 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 g�1 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 g�1 to-tal VS added from co-digestion of cattle manure and specified riskmaterials.

3.2. Methane concentration

Methane concentrations at OL20 for all M:WM ratios are shownin Fig. 3. Traces of methane gas (concentration: 0.2–2%) 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 final 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 L�1. This indicates that effective inactivation ofmethanogens in the microflora 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 for

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Fig. 3. Methane concentrations in the gas produced at organic loading of 20 gVS L�1. 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 L�1 as compared to 20 g VS L�1

(Table 1). The observed methane production at 20 g VS L�1 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 7–7.5 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 L�1) 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 significant(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 linear(P < 0.001) and quadratic relationships with respect to organicloading (P < 0.05). The maximum VS degradation (40.8%) wasachieved with M:WM 10:90 at OL60.

There were significant interactions between mixing ratio andorganic loading for both VFA production (P < 0.001) and overall in-crease in total VFA as a percentage of initial concentration(P < 0.001). Differences in mean VFA productions between OL20,OL40 and OL60 were significant for all M:WM ratios (P < 0.001) ex-cept M:WM 70:30 and 0:100 (P > 0.05). Similarly, the productionsdiffered (P < 0.05) between all M:WM ratios at OL20, OL40 andOL60. Acetate and butyrate were the most prevalent VFA in allthe treatments (Fig. 4). High productions of butyrate were ob-served in treatments with more waste milk than manure, suggest-ing that butyrate-type fermentation was dominant in digesterswith more waste milk as substrate. Distribution of VFA has beenobserved to be mostly determined by substrate types, with carbo-hydrate rich substrates usually take butyrate fermentation typeand protein rich substrates follow the acetate fermentation (Wanget al., 2011). Lactose in waste milk was probably utilized to pro-duce more butyrate than other VFAs. The observed phenomenonis also supported by findings of Shen et al. (1996), who reportedthat Butyribacterium methylotrophicum produced more butyratefrom lactate than from glucose. High production of butyrate coin-cided well with high hydrogen yield as observed in M:WM 50:50,30:70 and 10:90 treatments. These results agree with findings ofprevious studies on hydrogen production from co-digestion of dif-ferent substrates in batch experiments (Zhu et al., 2008; Wanget al., 2011). They reported that high butyrate production corre-lated with high hydrogen yield.

3.4. Total and cefazolin resistant bacteria

Bacterial load of mixed substrates at the three different organicloadings tested are presented in Fig. 5. Total bacteria reduction of0.5–3.3log cfu g�1 DM was observed in all the treatments at theend of the experiments. The overall reduction as a percentage ofinitial concentration showed a significant interaction betweenmixing ratio and organic loading (P < 0.05). The reductions differedsignificantly between M:WM ratios at OL20 (P < 0.001) and OL40(P < 0.001). Conversely, the mean reductions between OL20, OL40and OL60 were not significantly different (P > 0.05) for M:WMratios except M:WM 70:30 (P < 0.001).

Table 1VS degradation and characteristics of manure:waste milk (M:WM) substrate.

OLa M:WM VS degradation (%) Total VFA initial (g L�1) Total VFA final (g L�1) pH initial pH final

20 70:30 16.5 ± 0.6b 2.62 ± 0.03 4.74 ± 0.20 7.3 ± 0.0 6.1 ± 0.150:50 18.2 ± 0.8 1.84 ± 0.03 4.23 ± 0.45 7.3 ± 0.0 5.2 ± 0.130:70 16.2 ± 1.6 1.10 ± 0.01 3.52 ± 0.49 7.2 ± 0.0 4.9 ± 0.110:90 14.0 ± 1.8 0.39 ± 0.01 2.02 ± 0.56 7.1 ± 0.0 4.5 ± 0.10:100 11.7 ± 1.8 0.04 ± 0.00 0.43 ± 0.06 6.9 ± 0.0 4.7 ± 0.1

40 70:30 15.8 ± 0.6 5.29 ± 0.07 8.74 ± 0.11 6.4 ± 0.0 5.5 ± 0.050:50 18.1 ± 0.3 3.91 ± 0.03 8.75 ± 0.03 6.4 ± 0.0 5.2 ± 0.030:70 21.4 ± 0.3 2.41 ± 0.02 8.71 ± 0.15 6.5 ± 0.0 4.8 ± 0.010:90 5.6 ± 1.0 0.75 ± 0.05 2.00 ± 0.04 6.6 ± 0.0 4.6 ± 0.00:100 19.9 ± 1.7 0.02 ± 0.00 0.80 ± 0.02 6.7 ± 0.0 4.4 ± 0.0

60 70:30 12.7 ± 0.7 7.96 ± 0.07 10.66 ± 0.98 6.1 ± 0.0 5.5 ± 0.050:50 13.1 ± 0.6 5.83 ± 0.02 10.03 ± 0.12 6.1 ± 0.0 5.2 ± 0.030:70 19.3 ± 1.0 3.57 ± 0.03 9.23 ± 0.20 6.2 ± 0.0 4.7 ± 0.110:90 40.8 ± 1.6 1.48 ± 0.01 8.16 ± 0.43 6.2 ± 0.0 4.5 ± 0.00:100 25.4 ± 0.6 0.30 ± 0.01 0.93 ± 0.05 6.3 ± 0.0 4.8 ± 0.3

a Organic loading (g VS L�1).b Data in table are means ± standard error.

S.A. Lateef et al. / Bioresource Technology 110 (2012) 251–257 255

Cefazolin resistant bacteria were not detected in M:WM50:50at OL40 and M:WM 30:70at both OL20 and OL40 at theend of the experiments. In remaining treatments, the reductionsranged from 1.11 to 6.51log cfu g�1 DM (Fig. 5). The overall reduc-tion as a percentage of initial concentration was dependent uponmanure:waste milk (P < 0.05) and organic loading (P < 0.05), withno interaction between the two main effects (P > 0.05). The reduc-tion was significantly higher (P < 0.05) at high organic loading(OL40 and OL60) than at low organic loading (OL20), whereasincreasing the organic loading above 40 g VS L�1 was associatedwith non-significant increase in the level of reduction (P > 0.05).The level of reduction was similarly greater in M:WM 50:50,30:70 and 10:90 than 70:30 (P < 0.05).

One of the objectives of this study was to determine how con-ditions presented by manure waste milk ratio and different or-ganic loading in combination with thermophilic treatment affectthe reduction of cefazolin resistant bacteria. General reduction

a

c

Fig. 4. VFA concentration of mixed substrates at three different organic loading, (a) ac

of more one logarithmic unit (>90%) of cefazolin resistant bacteriawas observed in all the treatments, indicating that high tempera-ture coupled with 5-day treatment period caused high stress ofcefazolin resistant bacteria. The observed reduction was expected.Together with suitable exposure time, temperature is consideredto be the most important factor for microbial growth inhibitionin an anaerobic digestion environment (Sahlstrom, 2003), andby extension a critical factor in destruction and inactivation ofantibiotic resistant bacteria (Diehl and Lapara, 2010). Thermo-philic temperature causes greater reduction of antibiotic resistantbacteria than mesophilic temperature (Ghosh et al., 2009). The re-sults of this study are consistent with those of Diehl and Lapara(2010) where it was reported that thermophilic treatment(55 �C) reduced substantial quantities of tetracycline resistantdeterminants in municipal wastewater, thereby reducing thenumber of resistant bacteria being introduced into theenvironment.

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etate, (b) – butyrate and (c) – propionate. Values are means with standard error.

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Fig. 5. Bacterial load of mixed substrates at three different organic loading, (a) –total bacteria and (b) – cefazolin resistant bacteria (nd – not detected). Values aremeans with standard error.

256 S.A. Lateef et al. / Bioresource Technology 110 (2012) 251–257

The reduction of cefazolin resistant bacteria was higher at 40and 60 g VS L�1 than 20 g VS L�1. This is attributable to high con-centration/accumulation of VFA and more acidic pH at these organ-ic loadings. Although limited information is available on howanaerobic digestion technology and its associated operating condi-tions (such as pH and VFA) affect the quantities of antibiotics resis-tant bacteria (Diehl and Lapara, 2010), VFA concentration alone, orin combinations with pH, temperature, exposure time and the de-gree of sensitivity of specific types of microorganism is believed toimpact upon injury of microorganisms in anaerobic digestion(Abdul and Lloyd, 1985; Salsali et al., 2008). Initial VFA concentra-tions, VFA production and accumulation were higher at 40 and 60 gVS L�1 than 20 g VS L�1 (Table 1). These concentrations, in combi-nation with high acidic pH presented by the conditions, causedgreater reduction of cefazolin resistant bacteria. These resultsagree with the findings of Salsali et al. (2008). They reported thatat elevated temperature (55 �C), addition of high concentrations(1500–6000 mg L�1) of VFA (equimolar mixture of acetic, propionicand butyric acids) and adjusting the media pH to 4.5 substantiallyreduced concentrations of Clostridium perfringens significantly after24 h treatment period.

Co-digestion of cow manure with waste milk stimulated VFAproduction. The net production of VFA increased with increasedconcentration of waste milk in the mixture, resulting in moreacidic final pH in M:WM 50:50, 30:70 and 10:90 (Table 1). Thislikely account for higher reduction of cefazolin resistant bacteriaobserved for M:WM 50:50, 30:70 and 10:90 as compared toM:WM 70:30. The results, thus, suggest that using high substrateconcentration with higher concentration of waste milk duringthermophilic biohydrogen production will present conditions thatwill substantially reduce the concentration of cefazolin resistantbacteria.

The study clearly shows that inclusion of waste milk from mas-titic cow treated with antibiotic as a substrate in cow manure-based biohydrogen production using thermophilic anaerobicdigestion will enhance hydrogen production. It also shows thatanaerobic digestion under hydrogen-producing conditions wouldbe ideal candidate for treatment and disposal of waste milk fromcow treated with antibiotic. The resistant genes have not beenidentified in this study. Further studies, using advanced technique,are needed to identify genes that encode resistant to cefazolin andother antibiotics use in treatment of mastitis in dairy cows. Also,the concentration of antibiotic residue in the waste milk was notmeasured, although our results suggest that it did not drasticallyaffect hydrogen production. Future studies could investigate im-pact of antibiotic residues in waste milk on hydrogen production.Given that the rate of production of waste milk is far less than thatof manure, large-scale application of optimal ratio of manure andwaste milk reported here may be unfeasible. Nevertheless, thisdoes not render the report of the study irrelevant to practical appli-cations, especially when considering added benefit of treatmentand disposal of waste milk. It only warrants more research workthat should look into co-digestion of cow manure, waste milk (atratio that represents actual production rate) and other sugar-richwastes. Alternatively, immediate practical application could betwo-stage process, where waste milk is included as a substrate ata concentrations less than 30% of total VS in the first stage forhydrogen production and the first stage serves as a pretreatmentfor second stage (methane production).

4. Conclusions

The study showed that inclusion of waste milk as a co-substrateduring biohydrogen production from cow manure could improvehydrogen production. Optimal manure to waste milk ratio and or-ganic loading for biohydrogen production from co-digestion of cowmanure and waste milk was shown to be 30:70 (VS/VS) and 40 gVS L�1 respectively. The study also showed that aside from heat-shock pretreatment of manure (inoculum), low initial pH is alsoa critical factor in inhibition of methanogenesis. Thermophilicanaerobic biohydrogen production could be an ideal candidatefor treatment of milk from mastitic cow treated with antibiotics.

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