Tmw

La

b

a

ARR1A

KABOT

1

pytwnciilmtlc

1d

Chemical Engineering Journal 168 (2011) 249–254

Contents lists available at ScienceDirect

Chemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /ce j

he use of thermochemical and biological pretreatments to enhance organicatter hydrolysis and solubilization from organic fraction of municipal solidaste (OFMSW)

.A. Fdez.-Güelfoa,∗, C. Álvarez-Gallegoa, D. Salesb, L.I. Romeroa

Department of Chemical Engineering and Food Technology, Faculty of Science, University of Cadiz, Poligono Rio San Pedros/n, 11510 Puerto Real, Cadiz, SpainDepartment of Environmental Technologies, Faculty of Sea and Environmental Sciences, University of Cadiz, 11510 Puerto Real, Cadiz, Spain

r t i c l e i n f o

rticle history:eceived 15 October 2010eceived in revised form8 December 2010ccepted 22 December 2010

eywords:naerobic digestioniological pretreatmentrganic fraction of municipal solid wasteshermochemical pretreatment

a b s t r a c t

The introduction of the anaerobic digestion for the treatment of the organic fraction of municipal solidwaste (OFMSW) is currently of special interest. The main difficulty in the treatment of this waste fractionis its biotransformation, due to the complexity of organic material. Therefore, the first step must beits pretreatment for breaking complex molecules into simple monomers, to increase solubilization oforganic material and improve the efficiency of the anaerobic treatment in the second step. The hydrolysisstage is considered the rate-limiting step for the anaerobic digestion of solid wastes. Thus, in this paperthermochemical and biological pretreatments were performed to accelerate the hydrolytic processes bymeans of a fast organic matter solubilization of industrial OFMSW.

The thermochemical pretreatments were conducted in oxidizing and inert atmospheres and sodiumhydroxide was employed as an alkaline agent. On the other hand, the biological pretreatments were

performed using mature compost, fungus Aspergillus awamori and activated sludge from a conventionalWWTP as enzymatic agents.

The results from the thermochemical pretreatment indicate that the best conditions for organic mattersolubilization were 180 ◦C, 3 g NaOH/L and 3 bar. Increments of soluble chemical oxygen demand (COD)of approximately 246% can be achieved in these conditions. In the case of biological pretreatments, themature compost showed the maximum hydrolytic activity with an increase of COD of 51% for the lower

.5%, v

inoculation percentage (2

. Introduction

Anaerobic digestion can be an attractive option, both as a dis-osal route and as a source of alternative energy. In the last fewears, much effort has been made at introducing anaerobic diges-ion processes for treating the organic fraction of municipal solidaste (OFMSW). However, the main obstacle in spreading this tech-ology is the lower biodegradation rate of solid wastes (due to thehemical composition and structure of lignocellulosic materials)n comparison to liquid ones. Generally, the methanogenic stages the rate-limiting step of the anaerobic digestion processes ofiquid wastes. However, studies on the anaerobic digestion of pri-

ary sludge and organic complex substrates have concluded thathe hydrolysis of organic matter to soluble substrate is the rate-imiting step for solid waste degradation [1]. Therefore, physical,hemical or biological pretreatment methods (or their combina-

∗ Corresponding author. Tel.: +34 956016379; fax: +34 956016411.E-mail address: alberto.fdezguelfo@uca.es (L.A. Fdez.-Güelfo).

385-8947/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.cej.2010.12.074

/v).© 2011 Elsevier B.V. All rights reserved.

tion) are required, in order to reduce the rate of such a limitingstep.

Thus, in his work on cellulose fermentation, Pavlostathis [2]found a negligible accumulation of hydrolyzed products in the reac-tor and concluded that the conversion of cellulosic matter to solubleproducts was the rate-limiting step in the overall process. Lee andDonaldson [3] also observed a low concentration of soluble com-pounds in cellulose fermentation processes and thus, the hydrolyticstage was considered the limiting stage of the process as well. Bymeans of activity assays of soluble and insoluble fraction of slaugh-terhouse effluents (considered complex wastes for their ruminantremains and fat content), Galisteo et al. [4] determined that thehydrolysis of organic-complex material was also a limiting stage ofthe degradation process.

Hence, several methods have been developed to improve the

biodegradability of complex wastes through the application of dif-ferent types of pretreatments: physical, chemical, biological andcombinations of them [5–15]. For complex substrates such as ligno-cellulosic materials, pretreatment causes a deep modification in thestructure of complex material and a decrease in the degree of poly-

250 L.A. Fdez.-Güelfo et al. / Chemical Engineering Journal 168 (2011) 249–254

Table 1Physicochemical characterization of the industrial OFMSW.

Parameter Data

pH 7.98Density (kg/m3) 650Alkalinity (g CaCO3/L) 18.13Ammonia (g NH3-N/L) 0.79Total nitrogen (g/kg) 29.0TS (g/g sample) 0.71VS (g/g sample) 0.16DOC (mg/g) 11.90

maw

iwidwwp

2

2

coM(tIhpo

hvaa[

atfTntp

sb(dfai(a

Table 2Design of experiments.

Thermochemical pretreatments Biological pretreatments

Variable Value Inoculum percentage (%, v/v)

consisted in a pile of MSW and municipal WWTP anaerobicallydigested sludge (17:3) on a wet basis (10,889 kg of OFMSW mixedwith 993 kg of sludge). The inoculum characteristics are detailed inTable 3.

Table 3Features and composition of the compost pile in dry base.

Parameter Data

OFMSW (kg) 10.889Sludge (kg) 993pH 7.27NH4

+ (mg/g) 0.008Conductivity (mS/cm) 4.92Organic matter (%) 25.24Carbon (%) 14.63NO3

− (mg/g) 0.14N (%) 0.74

Total VFA (mgAcH/L) 32.65

erization, thus weakening the molecular bonds between ligninnd carbohydrates and increasing the superficial area of particulateastes.

This paper examines thermochemical alkaline and three biolog-cal pretreatments applied to organic fraction of municipal solid

aste (OFMSW) in order to enhance waste hydrolysis so as toncrease both the anaerobic biodegradability and overall degra-ation rate of the waste. The pretreatments mentioned aboveere expected to be particularly effective for industrial OFMSWith large-sized particles from a municipal solid waste treatmentlant.

. Materials and methods

.1. Methodology

Assays of thermochemical and biological pretreatments werearried out to accelerate the hydrolytic rate and increase the anaer-bic biodegradability of the OFMSW from an 880 t/d industrialSW treatment plant called Las calandrias in Jerez de la Frontera

Cadiz, Spain). The average particle size of the waste selected forhis study was 30 mm and its characterization is shown in Table 1.mportantly, the morphology of the waste used in the experimentsas not been altered in any way, i.e., the waste has not beenrocessed by drying or milling. The OFMSW had completely theriginal features of industrial waste.

The thermochemical pretreatments carried out used sodiumydroxide (NaOH) as an alkaline agent. In this case, the operativeariables to be studied were: temperature, pressure, NaOH dosagend type of atmosphere (inert (N2) or oxidizing (synthetic air))ccording to the experimental conditions reported in the literature16–21].

In the case of biological pretreatments, different enzymaticgents were tested: activated sludge from municipal and conven-ional WWTP placed in Puerto Real (Cadiz, Spain), mature compostrom the L. calandrias plant and finally, fungus Aspergillus awamori.his fungus has been elected since it is a variety of Aspergillusiger, biological-hydrolytic agent widely used and referenced inhe literature [6]. The operational variable in these assays was theercentage of inocula in terms of volume.

The effect of the pretreatments performed on organic matterolubilization has been measured in terms of dissolved organic car-on (DOC), dissolved volatile solids (DVS), total volatile fatty acidsTVFA) and soluble chemical oxygen demand (sCOD). TVFA wasetermined according to Riau et al. [22]. Individual C2–C7 volatileatty acids (VFA), including iC4, iC5 and iC6, were measured bygas chromatograph (Shimadzu GC-17 A) equipped with a flame

onization detector (FID) and a capillary column filled with Nukolpolyethylene glycol modified by nitroterephthalic acid). DVS, DOCnd sCOD were determined according to Standard Methods [23].

T (◦C) 120–150–1802.5, 5.0, 7.5 and 10Pressure (bar) 1–5–10

Dose of NaOH (g/L) 1–3–5

2.2. Thermochemical pretreatments

The experiment was conducted according to a 3n-type facto-rial design (3 being the number of the variable and “n” being thenumber of levels of each variable tested). The variables were tem-perature, pressure and alkaline agent dosage, as described in theplanning sheet shown in Table 2. The factorial design established27 experiments.

The assays were carried out in a 1 L non-stirred pressurevessel (ParrTM, series 4600-4620) equipped with two proportional-integral-derivative (PID) controllers for temperature and pressurefitting. The pressure was regulated by means of a pneumatic over-pressure valve activated by a compressor. A heating jacket was alsoemployed to warm the system.

Each reactor was filled to 60% of its capacity with OFMSW con-taining a total solids (TS) concentration of 30%. The alkaline agentwas applied by means of a 10 M-NaOH solution. The operation timewas 30 min in all cases established as optimal for this waste [24].

The inert and oxidizing atmospheres were achieved using N2and the air supply from a gas cylinder, respectively.

2.3. Biological pretreatments

Three biological agents (mature compost, the fungus A. awamoriand waste activated sludge) were used. The inocula concentrationsfor each of these were 2.5% (v/v); 5% (v/v); 7.5% (v/v) and 10% (v/v).In all cases, the operation time was 24 h established as optimal forbiological pretreatment with the same of OFMSW [24].

Four 1 L stainless steel reactors operating in batch mode withoutagitation were used to conduct the assays. The reactors had a topgas exit to evacuate the gases generated during the hydrolysis stage.

2.3.1. Mature compostThe reactors were loaded to 60% of their volume with

industrial OFMSW containing 30% of TS. The mature compost (den-sity = 0.58 g/mL) came from a windrow composting system, which

total

DOC (mg/g) 21.41P2O5 (mg/g) 0.26C/N ratio 19.7

L.A. Fdez.-Güelfo et al. / Chemical Engine

Table 4Microbiological characterization of the activated sludge.

Sample Microorganisms/mL % Viables % Non-viables

CTC sludge1 1.43E + 0.960.26 39.74

2

EoC

flfccw

2

Ostmtua

aGgms(tgT

3

3

s

3

Tir

ocioiaK5m

cw

DAPI sludge1 2.38E + 0.9

CTC sludge2 1.23E + 0.968.32 31.68DAPI sludge2 1.81E + 0.9

.3.2. A. awamori fungusA. awamori spores, stored at 4 ◦C, were provided by the Food

ngineering and Technology research group from the Departmentf Chemical Engineering and Food Technology of the University ofadiz.

The culture medium for their growth consisted in Agar–wheatour mixed to 5–2%, respectively. Five-day incubations were per-

ormed at 30 ◦C in aerobic conditions. After incubation, the sporeoncentrations in the inoculum were determined by microscopyount. Two counts were developed and the average result obtainedas 7.77E + 05 spores/mL.

.3.3. Activated sludge from municipal and conventional WWTPThe reactors were loaded to 60% of their volume with industrial

FMSW containing 30% of TS inoculated directly with activatedludge from a municipal and conventional WWTP, according tohe inoculation percentage described in Table 2. Epifluorescence

icroscopy through DAPI-fluorochrome was used to quantify theotal microorganisms in the activated sludge samples. The protocolsed [25] was in keeping with the protocol described by Kepnernd Pratt [26].

This technique was not capable of distinguishing between viablend total microorganisms and thus, the method developed byriebe and Nielsen [27] was used to determine the viable microor-anisms. This method is based on the use of epifluorescenceicroscopy to detect the formazen formed when the original sub-

trate (Tetrazolium salt: 5-cyano-2, 3-ditolyl tetrazolium chlorideCTC)) is reduced to CTC-formazen by the respiratory activity ofhe cells. The results obtained for the total and viable microor-anisms in the activated sludge used in the assay are shown inable 4.

. Results and discussion

.1. Thermochemical pretreatments

The solubilization yield was estimated as the difference betweenoluble organic matter at the beginning and the end of the test.

.1.1. Inert atmosphereThe solubilization yields obtained, expressed as DOC, DVS,

VFA and sCOD for the different runs (120, 150 and 180 ◦C) innert and oxidizing atmospheres (N2), are shown in Fig. 1a and bespectively.

As can be seen in Fig. 1a, the best solubilization yield in termsf DOC was obtained at higher temperatures when the operationalonditions corresponded with intermediate pressure (3 bar) andntermediate alkali dosage (3 g NaOH/L). Furthermore, pressuresver 5 bar and alkali dosages of over 3 g NaOH/L caused a signif-cant decrease in organic matter solubilization. The latter resultsre in line with the conclusions obtained by Penaud et al. [28] andim et al. [21], which confirmed that NaOH additions of more than

g NaOH/L did not lead to further significant increases of organicatter solubilization.When the temperature was increased to 120–180 ◦C, the effi-

iency of pretreatment was seen to rise; this finding is in accordanceith the findings established by Delgenés et al. [29] who reported

ering Journal 168 (2011) 249–254 251

that temperatures ranging between 90 and 160 ◦C cause an increasein organic matter solubilization. Thus, if the operational conditionsare 120 ◦C, 5 bar of pressure and 3 g NaOH/L and the tempera-ture is increased following the 150–180 ◦C sequence, solubilizationimproves. In these cases, the values for solubilization yields mea-sured as increments of DOC were 143.3% (120 ◦C, 5 bar, 3 g NaOH/L),153.13% (150 ◦C, 5 bar, 3 g NaOH/L) and 224.5% (180 ◦C, 5 bar, 3 gNaOH/L).

The above results may be associated to the fact that thehydrolytic capacity of the NaOH increases when the opera-tional temperature is increased. Nevertheless, several authors haveobserved a decrease in this synergic effect between NaOH and tem-peratures over 180 ◦C [29].

Thus, the best conditions for conducting a thermochemical pre-treatment of industrial OFMSW with NaOH are: a high temperature(180 ◦C), an intermediate dose of NaOH (3 g/L) and intermediatepressures (3 bar).

3.1.2. Oxidizer atmosphere (synthetic air)On the basis of the conclusions established in the previous sec-

tion and in order to study the influence of atmosphere type onsolubilization yield, the selected conditions in the runs at inertatmosphere were reproduced in an oxidizing atmosphere. Theresults are showed in Fig. 1a and b.

As can be seen in Fig. 1a, the best solubilization yield in aninert atmosphere, measured as increments of DOC, was obtainedat 180 ◦C, 5 bar and 3 g NaOH/L.

Furthermore, as can be seen in Fig. 1b, if the results obtainedfrom both types of atmospheres under the same operationalconditions are compared, decreased solubilization yields can beobserved in oxidizing conditions. Working pressure may solu-bilize part of the O2 contained in the oxidizing atmosphere tothe liquid phase. Hence, in aggressive temperature and pressureconditions, this oxidant agent may degrade a significant organicmatter fraction of the waste. In fact, another pretreatment com-monly reported in the literature uses ozone as the oxidizing agent[30].

Thus, under 180 ◦C, 5 bar and 3 g NaOH/L conditions, the sol-ubilization yield measured as increments of DOC, DVS, TVFA andsCOD fell to 58.90%, 80.98%, 35.37% and 141.90% respectively. Withrespect to the best run in inert atmosphere, this meant a decreasein the solubilization yield of 74% as DOC, 78% as DVS, 81% as TVFAand 42% as sCOD. The results obtained from applying these thermo-chemical pretreatments to OFMSW are very innovative and thus,these solubilization yields cannot be compared with those obtainedby other authors for the same type of waste.

3.2. Biological pretreatments

As in the thermochemical pretreatments, the solubilizationyield was estimated as the difference between soluble organic mat-ter at the beginning and the end of the test. However, in biologicalpretreatments, the soluble organic matter at the end of the assayis the net difference between two processes: solubilization andmetabolic consumption by the microbial population.

The results obtained in the runs corresponding to the threebiological agents tested are shown in Fig. 2. As can be seen, thesolubilization yield decreased when the inoculation percentagewas increased. This may be related to the increase in the concen-tration of active microorganisms associated with the increase inthe percentage of inoculation. For inoculation percentages higher

than 2.5% (v/v), the consumption of solubilized organic matter bymetabolic activity increases and, therefore, the net solubilizationyields decrease.

Thus, the best results in the organic matter solubilization toliquid phase were obtained for the lower inoculation percentage

252 L.A. Fdez.-Güelfo et al. / Chemical Engineering Journal 168 (2011) 249–254

F ochem( 180 ◦C

tttp

fatw

ls

ig. 1. (a) Solubilization yields of organic matter (in terms of DOC) applying thermin terms of sCOD, DVS, TVFA and DOC) applying thermochemical pretreatments at

ested (2.5%) in all the biological agents tested. The values of DOC,otal TVFA and sCOD present an analogous trend with regard tohe proportion of inoculum, which decreased when the inoculationercentage was increased.

Furthermore, in the tested conditions the best biological agentor the solubilization of organic matter to liquid phase, measureds increments of DVS, TVFA and sCOD, is the mature compost. Inhese conditions, the values corresponding to solubilization yields

ere: 41.67% in DVS, 22.43% in TVFA and 50.81% in sCOD.

In summary, the application of thermochemical and/or bio-ogical pretreatments to OFMSW enhances the hydrolysis andolubilization of organic matter.

Fig. 2. Comparison of solubilization yields for the different biol

ical pretreatments in inert atmosphere; (b) solubilization yields of organic matterand different atmospheres.

3.3. Statistical analysis

The statistical software SPSS® 15.0 was used for statistical pro-cessing of experimental results. First the Tukey test was applied.This test determines if experimental data has a normal distribution.So, if the value of significance (Sig) is greater than 0.05, experimen-tal data follows a normal distribution.

Subsequently, an analysis of variance (ANOVA) was applied

to determine if the data series presented statistical significantdifference. This analysis requires that all data follow a normal dis-tribution. For this reason, previously, Tukey test was performed.Similarly, following the analysis of variance, if the significance value

ogical agents according with the inoculation percentage.

L.A. Fdez.-Güelfo et al. / Chemical Engine

Table 5ANOVA analysis: influence of pressure and dose factors on each parameter whenthe temperature is fixed.

120 ◦C 150 ◦C 180 ◦C

Pressure Dose Pressure Dose Pressure Dose

�DOC 0.018 0.607 0.006 0.716 0.373 0.064�DVS 0.034 0.541 0.073 0.621 0.030 0.506�VFA 0.021 0.627 0.037 0.544 0.206 0.234�COD 0.098 0.320 0.089 0.544 0.352 0.109

Bold values indicate that pressure factor affects significantly.

Table 6ANOVA analysis: influence of temperature on each parameter when the pressure orNaOH dose is fixed.

Pressure(1 bar)

Dose(1 g/L)

Pressure(5 bar)

Dose(3 g/L)

Pressure(10 bar)

Dose(5 g/L)

�DOC 0.016 0.23 0.243 0.140 0.003 0.047�DVS 0.006 0.097 0.545 0.823 0.000 0.438�VFA 0.019 0.115 0.776 0.432 0.001 0.862�COD 0.029 0.397 0.803 0.798 0.169 0.360

Bold values indicate that temperature factor affects significantly.

Table 7ANOVA analysis: influence of biological agent and inoculation percentage on eachparameter.

Biological agent Inoculation percentage

�DOC 0.384 0.018�DVS 0.007 0.416

B

ib

3

fpt

stDtdi

3

iipl

4

cObNy

575–592.

�VFA 0.978 0.008�COD 0.071 0.263

old values indicate that factor affects significantly.

s less than 0.05 indicates that there are significant differencesetween the data.

.3.1. Thermochemical pretreatmentsAs can be seen in Table 5, when the temperature is set, the critical

actor in the performance of organic matter solubilization is theressure, since the NaOH dose did not significantly affect in any ofhe cases.

On the other hand, as can be seen in Table 6, when the pres-ure is set, the temperature factor has a significant influence onhe organic matter solubilization yield (expressed in terms of DOC,VS and VFA) at low (1 bar) and high (10 bar) pressure. Similarly,

he temperature factor has a significant influence at high NaOHose (5 g/L) when organic matter solubilization yield is expressed

n terms of DOC.

.3.2. Biological pretreatmentsAs can be seen in Table 7, the type of biological agent signif-

cantly affects on the organic matter solubilization yield when its expressed in terms of DVS. On the other hand, the inoculationercentage significantly influences on the organic matter solubi-

ization yield when it is expressed in terms of DOC and VFA.

. Conclusions

The results show that it was possible to increase soluble organicarbon by thermochemical and biological pretreatments of the

FMSW. The best result among the assays developed was obtainedy the thermochemical pretreatment at 180 ◦C, 5 bar and 3 gaOH/L in an inert atmosphere, which produced a solubilizationield of 224.5% in terms of sCOD.

ering Journal 168 (2011) 249–254 253

4.1. Thermochemical pretreatments

When the temperature is set, the best results are obtained atintermediate NaOH dose (3 g/L) and pressure (5 bar). Pretreatmentat 10 bar cause a reduction in the solubility of organic compounds,which can be related to the effect of waste compression. When thetemperature is changed, the efficiency increases as temperatureincreased, finding the best results at 180 ◦C. This could be linked tothe hydrolytic capacity of NaOH increases with temperature.

From the statistical point of view, the pressure factor affects sig-nificantly regardless of temperature. On the other hand, the NaOHdose does not affect significantly. Finally, when NaOH dose is set,statistical analysis indicates that the temperature factor affectssignificantly only at high dose (5 g/L). This behavior have beenobserved by several authors [28] due the synergic effect betweenNaOH adittion and temperatures over 180 ◦C. Therefore, it can beconcluded that pressure and temperature are the most importantfactors in the efficiency of solubilization of these pretreatments.

4.2. Biological pretreatments

As regards biological pretreatments, mature compost was thebest hydrolytic agent according to the increase of organic matter;the optimum inoculation percentage for this study was 2.5% (v/v).The solubilization yield measured as an increment of sCOD withthis inoculation percentage was 50.81%.

4.3. Comparison between thermochemical and biologicalpretreatments

Thermochemical pretreatments in this study have showed animportant improvement degree on solubilization (50–250%) com-pared to biological pretreatments (10–50%), next to 5 times greater.

However, from the point of view of industrial applicability, itis important to emphasize that thermochemical pretreatments arein economic disadvantage versus usual biological pretreatmentsdue to mainly reagent and power consumption costs associatedwith the first one. The main advantage of biological pretreatment isthat implementation on industrial composting and biomethaniza-tion plants do not require expensive additional facilites or complexmodifications of the process (just a pretreatment zone on head ofthe process) with no special safety considerations. The biologicalagent could be mature compost obtained as final solid product ofthe plant.

Further evaluation studies, including economical and perfor-mance topics, are necessary for well-argued selection.

Acknowledgements

This work was supported by the Spanish Ministry of Scienceand Innovation (Project CTM2007-62164/TECNO), the Innovation,Science and Enterprise Department of the Andalusian Government(Project P07-TEP-02472) and the European Regional DevelopmentFund (ERDF).

References

[1] P. Chulhwan, L. Chunyeon, K. Sangyong, Ch. Yu, C.H. Howard, Upgrading ofanaerobic digestion by incorporating two different hydrolysis processes, J.Biosci. Bioeng. 100 (2) (2005) 164–167.

[2] S.G. Pavlostathis, Preliminary conversion mechanisms in anaerobic digestionof biological sludge, J. Environ. Eng. Div. Proc. Am. Soc. Civ. Eng. 114 (1988)

[3] D. Lee, T.L. Donaldson, Anaerobic digestion of cellulosic wastes, Biotechnol.Bioeng. Symp. 14 (1984) 503–505.

[4] M. Galisteo, M. Mallo, J. Martínez, Degradabilidad anaerobia de efluentes com-plejos, in: Proceeding Fifth Latin-American Workshop-Seminar. WastewaterAnaerobic Treatment, Vinas del Mar. Chile, 1998.

2 ngine

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

54 L.A. Fdez.-Güelfo et al. / Chemical E

[5] H. Carrère, C. Dumas, A. Battimelli, D.J. Batstone, J.P. Delgenès, J.P. Steyer, I.Ferrer, Pretreatment methods to improve sludge anaerobic degradability: areview, J. Hazard. Mater. 183 (2010) 1–15.

[6] L. Coulibaly, G. Gourene, N. Agathos, Utilization of fungi for biotreatment of rawwastewaters, Afr. J. Biotechnol. 2 (2003) 620–630.

[7] A.T.W.M. Hendriks, G. Zeeman, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresour. Technol. 100 (2009)10–18.

[8] M. López Torres, Ma.del.C. Espinosa Lloréns, Effect of alkaline pretreat-ment on anaerobic digestion of solid wastes, Waste Manage. 28 (11) (2008)2229–2234.

[9] K. Hwang, E. Shin, H. Choi, A mechanical pre-treatment of waste activatedsludge for improvement of anaerobic digestion system, Water Sci. Technol. 36(12) (1997) 111–116.

10] R. Rodríguez-Vázquez, G. Villanueva-Ventura, E. Rios-Leal, Sugarcane Bagassepith dry pre-treatment for single cell protein production, Bioresour. Technol.39 (1992) 17–22.

11] M. Ropars, R. Marchal, J. Pourquié, J.P. Vandecasteele, Large-scale enzymatichydrolysis of agricultural lignocellulosic biomass Part 1: pre-treatment proce-dures, Bioresour. Technol. 42 (1992) 197–204.

12] H. Rughoonundun, C. Granda, R. Mohee, M.T. Holtzapple, Effect of thermo-chemical pretreatment on sewage sludge and its impact on carboxylic acidsproduction, Waste Manage. 30 (2010) 1614–1621.

13] H. Wang, Ho. Wang, W. Lu, Y. Zhao, Digestibility improvement of sorted wastewith alkaline hydrothermal pretreatment, Tsinghua Sci. Technol. 14 (3) (2009)378–382.

14] Ch. Ying-Chih, Ch. Cheng-Nam, L. Jih-Gaw, H. Shwu-Jiuan, Alkaline and ultra-sonic pre-treatment of sludge before anaerobic digestion, Water Sci. Technol.36 (11) (1997) 155–162.

15] J. Zhu, C. Wan, Y. Li, Enhanced solid-state anaerobic digestion of corn stover byalkaline pretreatment, Bioresour. Technol. 101 (2010) 7523–7528.

16] S. Sawayama, S. Inoue, K. Tsukahara, T. Ogi, Thermochemical liquidization ofanaerobically digested and dewatered sewage sludge, Bioresour. Technol. 55(1996) 141–144.

17] S. Sawayama, S. Inoue, T. Minowa, K. Tsukahara, T. Ogi, Thermochemical liq-uidization and anaerobic treatment of kitchen garbage, J. Ferment. Bioeng. 5(1997) 451–455.

[

ering Journal 168 (2011) 249–254

18] K. Tsukahara, T. Yaguishita, T. Ogi, S. Sawayama, Treatment of liquid fractionseparated from liquidized food waste in an upflow anaerobic sludge blanketreactor, J. Biosci. Bioeng. 4 (1999) 554–556.

19] Q. Wang, M. Kuninobu, H. Ogawa, Y. Kato, Degradation of volatile fatty acids inhighly efficient anaerobic digestion, Biomass Bioenergy 16 (1999) 407–416.

20] C. Lin, Y. Lee, Effect of thermal and chemical pretreatments on anaerobic ammo-nium removal in treating septage using the UASB system, Bioresour. Technol.83 (2002) 259–261.

21] J. Kim, C. Park, T. Kim, M. Lee, S. Kim, J. Lee, Effects of various pretreatments forenhanced anaerobic digestion with waste activated sludge, J. Biosci. Bioeng. 3(2003) 271–275.

22] V. Riau, M.A. De la Rubia, M. Pérez, Temperature-phased anaerobic digestion(TPAD) to obtain class A biosolids. A discontinuous study, Bioresour. Technol.101 (2010) 65–70.

23] APHA, AWWA & WEF, Standard Methods for the Examination of Water andWastewater. 19th ed. (1995), Washington, DC, New York, USA.

24] Romero-Aguilar, M.A. Thermochemical and biological pretreatments appliedto OFMSW before the acidogenic anaerobic digestion. Research work to obtainAdvanced Studies Certificate. (2008).

25] R. Solera, L.I. Romero, D. Sales, Determination of the microbial population inthermophilic anaerobic reactor: comparative analysis by different countingmethods, Anaerobe 7 (2) (2001) 79–86.

26] R.L. Kepner, J.R. Pratt, Use of fluorochromes for direct enumeration of totalbacteria in environmental samples: past and present, Microbiol. Rev. 58 (4)(1994) 603–615.

27] T. Griebe, P.H. Nielsen, Enzymatic activity in the activated-sludge floc matrix,Appl. Microbiol. Biotechnol. 43 (1995) 755–761.

28] V. Penaud, J.P. Delgenés, R. Moletta, Thermo-chemical pretreatment of a micro-bial biomass: influence of sodium hydroxide addition on solubilization andanaerobie biodegradability, Enzyme Microb. Technol. 25 (1999) 258–262.

29] J.P. Delgenés, V. Penaud, M. Torrijos, R. Moletta, Investigation on the changes in

anaerobic biodegradability and biotoxicity of an industrial microbial biomassinduced by thermochemical pre-treatment, Water Sci. Technol. 41 (3) (2000)137–144.

30] C. Bougrier, A. Battimelli, J.-P. Delgenes, H. Carrere, Combined ozone pretreat-ment and anaerobic digestion for the reduction of biological sludge productionin wastewater treatment, Ozone: Sci. Eng. 29 (2007) 201–206.