Co-gasification of sewage sludge and woody biomass in a fixed-bed downdraft gasifier: Toxicity assessment of solid residues

HepG2 liver cell and MRC-5 lung fibroblast were used to study the toxicity of the bottom ash as the toxins in the bottom ash may enter blood circulation by drinking the contaminated water

Co-gasification of sewage sludge and woody biomass in a fixed-bed

downdraft gasifier: Toxicity assessment of solid residues

Le Ronga, Thawatchai Maneerunga, Jingwen Charmaine Ngb, Koon Gee Neohb,⇑, Boon Huat Bayc,

a

NUS Environmental Research Institute, National University of Singapore, 1 Create Way, Create Tower #15-02, Singapore 138602, Singapore

b Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore

c

Department of Anatomy, Yong Loo Lin School of Medicine, National University Health System, 4 Medical Drive, Singapore 117597, Singapore

d

School of Mechanical Engineering, Shanghai Jiaotong University, Shanghai 200240, China

Article history:

Received 1 August 2014

Accepted 28 November 2014

Available online 19 December 2014

Keywords:

Gasification

Sewage sludge

Bottom ash

Downdraft gasifier

Toxicity assessment

a b s t r a c t

As the demand for fossil fuels and biofuels increases, the volume of ash generated will correspondingly increase Even though ash disposal is now strictly regulated in many countries, the increasing volume of ash puts pressure on landfill sites with regard to cost, capacity and maintenance In addition, the prob-ability of environmental pollution from leakage of bottom ash leachate also increases The main aim of this research is to investigate the toxicity of bottom ash, which is an unavoidable solid residue arising from biomass gasification, on human cells in vitro Two human cell lines i.e HepG2 (liver cell) and MRC-5 (lung fibroblast) were used to study the toxicity of the bottom ash as the toxins in the bottom ash may enter blood circulation by drinking the contaminated water or eating the food grown in bottom ash-contaminated water/soil and the toxic compounds may be carried all over the human body including

to important organs such as lung, liver, kidney, and heart It was found that the bottom ash extract has a high basicity (pH = 9.8–12.2) and a high ionic strength, due to the presence of alkali and alkaline earth metals e.g K, Na, Ca and Mg Moreover, it also contains concentrations of heavy metals (e.g Zn, Co, Cu,

Fe, Mn, Ni and Mo) and non-toxic organic compounds Although human beings require these trace ele-ments, excessive levels can be damaging to the body From the analyses of cell viability (using MTS assay) and morphology (using fluorescence microscope), the high toxicity of the gasification bottom ash extract could be related to effects of high ionic strength, heavy metals or a combination of these two effects Therefore, our results suggest that the improper disposal of the bottom ash wastes arising from gasifica-tion can create potential risks to human health and, thus, it has become a matter of urgency to find alter-native options for the disposal of bottom ash wastes

Ó 2014 Elsevier Ltd All rights reserved

1 Introduction

Thermal processes such as incineration and gasification can be

considered as sustainable technology which is mostly used for

the solid waste treatment It is sustainable both in terms of waste

volume reduction and a source of renewable energy However,

burning of solid waste in incinerators leads to serious negative

environmental and human health effects, due to emission of

mas-sive amount of toxic gases (Beylot and Villeneuve, 2013; Roy et al.,

2011) resulting from the complete combustion process inside the

incinerators Gasification is a process that slowly converts large

molecules of organic or fossil based carbonaceous materials into smaller molecular and finally, into gaseous product which mainly consists of carbon monoxide, hydrogen and carbon dioxide (Ruiz

et al., 2013; Simone et al., 2012) This is achieved by chemically reacting carbonaceous materials at high temperatures (>700 °C) with a controlled amount of oxygen and/or steam to avoid com-plete combustion (Samolada and Zabaniotou, 2014;Zhang et al.,

2013) Therefore, gasification not only provides a cost-effective and environmental friendly way of discharging solid waste, but also produces syngas as a clean energy fuel, offering an alternative clean process for recovering energy from the waste

Apart from the gaseous product, gasification also generates solid residues as unavoidable by-products, which amounts varying from 10% to 30% of the original feedstock mass, depending on effi-ciency of the gasifier and feedstock materials (Sabbas et al., 2003; Belgiorno et al., 2003) Depending on the carbon content of the http://dx.doi.org/10.1016/j.wasman.2014.11.026

0956-053X/Ó 2014 Elsevier Ltd All rights reserved.

⇑ Corresponding authors Tel.: +65 65162176; fax: +65 67791936 (K.G Neoh).

Tel.: +65 65165079; fax: +65 67791936 (C.H Wang).

E-mail addresses: chenkg@nus.edu.sg (K.G Neoh), chewch@nus.edu.sg

(C.-H Wang).

Contents lists available atScienceDirect

Waste Management

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / w a s m a n

residue, it can be classified as char (unreformed carbon) or bottom

ash (primarily minerals and metals with minimal carbon) Char is

usually re-introduced into the gasifier to generate more energy

On the other hand, bottom ash is generally disposed in landfills

(Travar et al., 2009) or re-utilized as a filling material for

construc-tion applicaconstruc-tions (Forteza et al., 2004) Although bottom ash is

clas-sified as nonhazardous waste, fresh bottom ash contains various

inorganic compounds, consisting mainly of oxides, hydroxides

and alkali salts, which cause a high pH value of the bottom ash

(Rocca et al., 2012) and trace amounts of heavy metals Moreover,

the fresh bottom ash may also contain organic compounds arising

from incomplete combustion of solid wastes (Liu et al., 2008) As a

result, the fresh bottom ash arising from solid waste gasification

can have toxic properties related to trace elements, organic

con-taminants and alkalinity, or a combination of these factors The

improper disposal of bottom ash wastes may cause all types of

pol-lution i.e air, soil, and water (Sivula et al 2012a; Sivula et al

2012b; Liu et al 2008)

As the demand for bioenergy production increases, ash and

res-idue volumes will increase Even though the ash disposal is now

strictly regulated in many countries, the rapid increase of ash

results in the high pressure on landfill sites regarding cost, capacity

and maintenance Moreover, ash leachate may leak or spill from

the landfill sites For example, in the Kingston Fossil Plant

(Tennes-see, USA) spill in 2008, 1.1 billion gallons (4,200,000 m3) of fly ash

slurry was spilled from an on-site landfill, covering more than

300 acres of surrounding land and water (Dewan, 2008) Therefore,

the chance of environmental pollution by ash leachate is ever

increasing The ash contaminants can accumulate inside plants

and animals, which can harm or kill them

Up to date the effect of emissions from the gasification of solid

wastes such as sewage sludge on the human health has seldom

been addressed (Kabir and Kumar, 2012; Pa et al 2011), especially

with regard to the effect of solid residues, because their effect may

last for a long period, such as 100–1000 years However, it is quite

difficult to draw firm conclusions on human toxicity, because even

the best emissions data is incomplete and the true impacts of most

chemicals and mixtures of chemicals are poorly understood

From those points of view, the main purpose of this work is to

investigate the toxicity of bottom ash arising from biomass and/or

solid waste gasification The main reason is that the bottom ash

arising from gasification is usually disposed in the landfills, and

the leaching of toxic compounds by rain or surface water can cause

severe pollution to environment and possibly endanger human

health By drinking the contaminated water or eating the food

grown in bottom ash-contaminated water/soil, toxic compounds

may be carried all over the human body to important organs such

as the lung, liver, kidney and heart Moreover, understanding of

toxic properties of bottom ash wastes generated from gasification

is necessary for establishing environmentally and economically

benign solid wastes management In this work, the extracts from

the bottom ashes arising from gasification processes of (i) pure

woodchip (which is represented by PWG in the following) and

(ii) 20 wt.% sewage sludge and 80 wt.% woodchips (which is

repre-sented by 20SWCG in the following) (all samples were collected

from our present work) were characterized by determination of

elements (i.e heavy metals, alkaline earth metals and alkali

met-als), polycyclic aromatic hydrocarbons (PAHs) and other organic

compounds concentrations Two types of human cell lines, i.e

HepG2 (liver cell) and MRC-5 (lung cell), were used to examine

toxicity of the bottom ash leachates The liver cell line, HepG2

was selected as it possesses a number of characteristic enzyme

pathways of human hepatocytes and is regarded as a ‘‘gold

stan-dard’’ in toxicology (Kang et al 2010; Babich et al 1988) MRC 5

lung fibroblasts were chosen as the lungs are major organs in the

respiratory system and as a large volume of blood passes through

the pulmonary circulation, toxic substances which may exert dele-terious effects are easily conveyed to the lungs The viability and morphology of HepG2 and MRC-5 cells were used to analyze the toxic effects on those human cells

2 Materials and methods 2.1 Collection of sewage sludge and bottom ash samples from gasification

Sewage sludge samples were obtained from the Ulu Pandan Water Reclamation Plant, under the Public Utilities Board (PUB), the National Water Agency of Singapore The sewage sludge sam-ple from different batches was mixed, dried and ground into pow-ders In the present study, woody biomass with an average length

of 35 mm, width of 10 mm, and thickness of 2.5 mm or its mixture with the above sewage sludge sample was used as the feedstock for gasification in a 10 kW fixed bed downdraft gasifier (from All Power Labs, USA) The air flow rate investigated was from 4.0 to 10.0 L/s The temperatures in pyrolysis and reduction zones were 550–800 °C and 650–900 °C, respectively The main processes tak-ing place in this downdraft gasifier can be described as follows:

 Drying  removal of moisture from the feedstock

 Pyrolysis  thermal breakdown the feedstock into tar and charcoal

 Combustion and tar cracking  burning of charcoal and tar to provide heat for the rest of the processes and the thermal crack-ing of a portion of the tar into CO and H2(syngas)

Ccharcoal;tar þ O2ðfrom airÞ ! CO2ðgÞ þ H2OðgÞ þ heat

 Reduction  reaction of combustion products and charcoal to produce syngas

CO2þ Ccharcoalþ heat ! 2CO

Within the gasifier the feedstock flowed downwards by gravity and air as a gasifying (or oxidizing) gas was introduced into the combustion zone of the reactor Bottom ash samples were col-lected from the ash pit at the bottom of the fixed bed downdraft gasifier after the gasification of pure woodchip (PWG) and 20% sludge and 80% woodchip (20SWCG) However, due to the struc-ture of the gasifier, some small char particles formed in the reduc-tion zone may fall into the ash pit and subsequently be mixed with the bottom ash As char can be recycled rather than landfilled in actual gasification processes, they were removed via sieving before the toxicity test

2.2 Extraction of soluble toxic substances from samples

Fig 1shows the process for solute extraction and toxicity test

8 g of sample of either sewage sludge or sieved bottom ash was weighed and placed in a 50 mL tube, and mixed with 40 mL of deionized (DI) water, resulting in a liquid-to-solid (L/S) ratio of 5.0 The mixture was then vortexed for 5 min, followed by over-night static leaching for 12 h, and centrifuged at 15,000 rpm for

10 min The supernatant was transferred into a separate tube and

the above centrifugation step was repeated twice to remove as

much particles as possible from the extracted solution The

col-lected supernatant was then filtered using a filter with a pore size

of 0.2lm (Minisart, Sartorius AG, Germany) This not only removes

the tiny particles originally in the sample but also helps to sterilize

the exaction solution by removing almost all of the bacteria Based

on experience, the filtration method is a more effective method for

disinfection than UV irradiation of the sample powders Since the

bottom ash extract was highly basic (as discussed below), a

neu-tralization step was adopted to avoid possible denaturation of

pro-teins in the cell culture medium This was done by gradually

adding 0.3 M nitric acid into the extract until its pH value reached

8, close to the pH value of the cell culture medium used, during

which no formation of any precipitates was observed This implies

that the amount of dissolved metals in the leachate did not change

substantially, which is consistent with the findings byMeima and

Comans (1997, 1999) The neutralization step was not needed for

extraction of sewage sludge since the solution had a pH value of

about 8.4 After the neutralization, the extract was freeze-dried

and the solute powder was then re-dissolved in cell culture

med-ium to the original volume (30 mL) before freeze-drying The

as-obtained solution, with designated solute concentration of C0,

was diluted with fresh medium to prepare extract solutions of

dif-ferent concentrations, such as C/C0= 0.5, 0.25, etc

2.3 Characterizations of the bottom ashes and their extractions

2.3.1 Scanning electron microscopy (SEM) analysis

Morphology of the bottom ashes arising from PWG and

20SWCG gasification was investigated by Scanning Electron

Microscopy (SEM, JEOL2872), carried out using an electron beam

of 15 kV with magnifications in the range of 2000–5000

2.3.2 X-ray diffraction analysis

Crystalline phases of bottom ashes arising from PWG and

20SWCG gasification were characterized by powder X-ray

diffrac-tion using SHIMADZU XRD-600 diffractometer with Cu Ka

radia-tion (k = 0.154 nm), operated at 40 kV and 30 mA Data was

collected at 0.02° with 5 s per step, in the 2h range of 20–50°

2.3.3 Elemental analysis

The elemental analyzer (Elementar Vario Micro Cube) was used

to determine the content of carbon (C), hydrogen (H), nitrogen (N)

and sulfur (S) in the feedstock materials The percentage by mass of

C, H, N and S was determined and the oxygen (O) mass percent was calculated by difference

2.3.4 Inductively Coupled Plasma (ICP) analysis Inductively coupled plasma analysis optical emission spectros-copy (ICP-OES) was employed for the detection of trace metals present in the feedstock, bottom ash product and its respective cell culture medium extracts Dual-view Optima 5300 DV ICP-OES sys-tem was used, which has a minimum detection level of 0.1 ppm Each sample was digested with HNO3/HCl and topped up to

10 ml with H2O before being analyzed The metals chosen for detection were Ti, Ag, Zn, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Al, Hg, As and Mo for bottom ashes and their cell culture medium extracts

2.3.5 pH value and conductivity measurement The pH value of each extract solution was measured using a Lab

850 pH meter (SCHOTT Instruments GmbH, Mainz, Germany) The conductivity of solution was measured using a Thermo Scientific™ Orion™ 3-Star Plus Conductivity Benchtop Meter (Thermo Fisher Scientific, MA, USA) Each measurement was repeated with three samples

2.3.6 Gas chromatography–mass spectrometry (GC–MS) The analysis of PAHs in the extracts was carried out using gas chromatography coupled with mass spectrometry (GC–MS) analysis (Shimadzu GCMS-QP2010 Plus with AOC-5000 auto injector with an Agilent J&W DB-5 ms column) Bottom ash samples were directly extracted using acetonitrile (ACN), with the same method as the cell culture extractions A mixture of methanol (MeOH) and dichloro-methane (DCM) in volume ratio 1:1 was used to further extract organic compounds from the aqueous cell culture extracts The fol-lowing conditions were used for the GC–MS analysis: Constant flow

of helium carrier gas at 45 cm/s; inlet at 300 °C, 40 psi until 0.2 min and purge flow 30 ml/min at 0.75 min; oven temperature between

55 °C to 320 °C at 25 °C/min and held 3 min; and a scan range of 50–350 g/mol A standard solution, M-610A, containing a mixture

of the 16 priority PAHs (AccuStandard, New Haven, USA) (including Acenaphthene, Anthracene, Benzo(a)anthracene, Fluoranthene, Naphthalene, Phenanthrene, Benzo(a)pyrene, Benzo(b)fluoranth-ene, Benzo(ghi)perylBenzo(b)fluoranth-ene, AcenaphthylBenzo(b)fluoranth-ene, Benzo(k)fluoranthBenzo(b)fluoranth-ene, Dibenzo(a,h)anthracene, Indeno(1,2,3-cd)pyrene, Chrysene, Fluo-rene and PyFluo-rene) was used as calibration standard in GC–MS analy-sis Post-run analysis of chemical components of each sample was carried out using the NIST library in the system

Fig 1 Schematic drawing of extraction process and in vitro toxicity test.

2.4 Culture of MRC-5 lung fibroblasts and HepG2 cells in 96-well plate

MRC-5 lung fibroblasts (from ATCC, USA) cultured in a cell

cul-ture flask, were harvested using trypsin and re-suspended in

med-ium (RPMI + 10% FBS + 1% Pen strep) to form a cell suspension This cell suspension was added into the wells of a 96-well with a total cell number of about 5000 cells in each well The well plate was then incubated at 37 °C and 5% CO2for 24 h, by which time the cells had fully attached to the bottom of the wells HepG2 cells (from ATCC, USA) were cultured in DMEM supplemented with10% FBS and 1% Pen strep A seeding density corresponding to about

5000 cells per well was used to avoid over-confluence

2.5 Toxicity evaluation of bottom ash extracts on human cells After the cells have attached, the original medium was replaced with 200lL of extract solution to examine the possible toxic effects on the human cells Four wells were used for each sludge extract concentration When the target time point was reached,

(b) (a)

Bottom ash Char

Fig 2 SEM photographs of (a) char and (b) bottom ash arising from gasification process.

Table 1

Elemental analysis of bottom ashes obtained from gasification of PWG and 20SWCG.

Bottom ash from PWG > 500lm 68.96 2.16 1.25 <0.50

Bottom ash from PWG < 500lm 26.03 0.94 <0.50 0.62

Bottom ash from 20SWCG > 500lm 53.91 1.93 <0.50 1.24

Bottom ash from 20SWCG < 500lm 19.92 1.09 <0.50 1.97

Note: PWG stands for pure wood chips gasification; 20SWCG stands for 20 wt.%

sewage sludge and 80 wt.% wood chips co-gasification.

Table 2

The pH values and concentration of alkali and heavy metals of bottom ashes and the extracts of bottom ashes obtained from gasification of PWG and 20SWCG.

<0.1 a

<0.1 a

<0.1 a

<0.1 a

<0.1 a

<0.1 a

<0.1 a

a

the plate for the morphology study was stained with LIVE/DEADÒ

Kits for mammalian cells (Invitrogen™, Life Technologies Pte Ltd,

Singapore) and observed under an Axio Observer fluorescence

microscope (Carl Zeiss, Germany) to check the cell morphology in

different wells At the same time, the medium in wells for viability

measurement was removed and replaced with a mixture of 100lL

of medium and 20lL of MTS solution (Promega, USA) According to the protocol suggested by the manufacturer, the plate was then incubated at 37 °C in a humidified 5% CO2 atmosphere for 3 h before the absorbance was read using the Tecan Genios MicroPlate reader at a wavelength of 490 nm and analyzed using software GraphPad Prism 4.0 Cell viability in each well was calculated as the ratio of the absorbance (which has a linear relationship with the number of live cells) of that well to that of the control well

2.6 Toxicity assessment of heavy metals on HepG2 cells

In order to confirm the possible toxic effects of heavy metals, soluble salts of metals Zn, Co, Cu, Fe and Mn were separately spiked into fresh cell culture medium to form solutions with the same metal concentration as that found in the extract of bottom ash from 20SWCG (i.e 5.37, 19.51, 4.19, 8.70 and 19.51 ppm for Zn,

Co, Cu, Fe and Mn, respectively) HepG2 cells were then incubated

in these solutions These solutions were also diluted to different concentrations and their cytotoxicity on HepG2 cells was studied Lastly, salts of the five metals at the same concentration as above were combined and added to culture medium The medium was then diluted and applied to HepG2 cells to check the combined effects on cell viability

2.7 Statistical analysis Software GraphPad Prism 4.0 was used for statistical analysis For cell viability in extracts of sewage sludge and bottom ash, the analysis was performed using one-way ANOVA, followed by a post hoc Tukey’s multiple comparison tests A value of p < 0.05 (which is the predetermined significance level) indicates the differ-ence in cell viability between the tested extract solution and the control is significant

0

500

1000

1500

20SWCG Bottom Ash

Cc

Fs

En Sp Hm Fe Qz

Mm

0

500

1000

1500

20 25 30 35 40 45 50

2 Theta (Degree)

PWG Bottom Ash

Cc

Sp

En Qz

Fs

Mm

Fig 3 XRD patterns of bottom ashes obtained from gasification of PWG and

20SWCG [(Cc) Calcite, CaCO 3 ; (Sp) Spurrite, Ca 5 (SiO 4 ) 2 CO 3 ; (En) enstatite, MgSiO 3 ;

(Qz) Quartz, SiO 2 ; (Fs) Ferrosilite, (Fe,Mg)SiO 3 ; (Fe) Metallic iron, Fe 0

; (Mm) Maghemite,c-Fe 2 O 3 ; (Hm) Hematile,a-Fe 2 O 3 ].

0.0

0.5

1.0

1.5

2.0

2.5

(x1,000,000)

Retention time (minutes)

(c)

1.0

2.0

3.0

4.0

5.0

(x1,000,000)

(b)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

(x1,000,000)

(a)

Fig 4 GC–MS analysis of PAHs in (a) standard M-610A with the concentration of

Table 3 Organic compounds which were found in different extracts of bottom ashes by GC– MS.

Benzene, 1,3-bis(1,1-dimethylethyl)- Benzene A, B, D Pentanedioic acid, 2,4-dimethyl-, dimethyl ester Carboxylic

acid

A

acid

D 9-Octadecenoic acid, 12-(acetyloxy)-, methyl ester,

Note: (A) and (B) are the ACN and MeOH/DCM extract of bottom ash from 20SWCG (C) and (D) are the ACN and MeOH/DCM extract of bottom ash from PWG.

3 Results and discussion

3.1 Physical morphology of solid residues

Fig 2shows the SEM images characterizing the physical

mor-phology of solid residues arising from our previous co-gasification

of sewage sludge and woody biomass which can be classified into

(i) bottom ash, which mainly contains inorganic compounds and

(ii) char, which is unreformed carbon Although char is usually

re-used as a feedstock for gasification, its highly porous structure

(as shown inFig 2a) make it become the excellent source for

pro-ducing of absorbent materials (such as activated carbon) that will

be studied in our future work On the other hand,Fig 2b shows that bottom ash mainly consists of the unreformed carbon and other inorganic compounds whose chemical composition will be discussed in the following sections

3.2 Chemical properties of bottom ashes 3.2.1 Elemental analysis of bottom ashes

Table 1shows the elemental analysis of bottom ash from pure woodchip gasification (PWG) and 20% sludge/woodchip co-gasifi-cation (20SWCG) with the different ranges of particle size obtained after sieving It can be seen that both PWG and 20SWCG bottom ashes with the particle size of higher than 500lm have high car-bon content (>50 wt.%) This result suggests that these two bottom ashes should be re-used as a feedstock for gasification in order to recover more energy Therefore, only the bottom ashes with the particle size lower than 500lm were considered as the bottom ash wastes and used for the following studies

3.2.2 ICP elemental analysis of bottom ashes The concentrations of alkali, alkaline earth and heavy metals of the bottom ashes and their extractions measured by ICP-OES are presented in Table 2 The results clearly show that both PWG and 20SWCG bottom ashes mainly contain alkali and alkaline earth metals, i.e K, Na, Ca, and Mg as the major components, which con-tributes to the high basicity (pH valves ranging from 9.2 to 13.4) of these two bottom ashes This result is in good agreement with the data reported in previous studies regarding ash originated from wood gasification (Gori et al., 2011; Tafur-Marinos et al., 2014)

0.0

0.5

1.0

1.5

2.0

Concentration (C/C 0 )

0 (control) 10 10 10 10 10 10 1

MRC-5 Cells

*

Fig 5 Measured absorbance in MTS assay of MRC-5 cells after exposure to sludge

extract for 24 h (C 0 is the concentration of the original sludge extract in the

medium) ( ⁄

p < 0.05 with respect to absorbance of control).

0.0

0.5

1.0

1.5

Concentration (C/C 0 )

HepG2 Cells 24 hr

(a)

*

0.0

0.5

1.0

1.5

Concentration (C/C 0 )

HepG2 Cells 48 hr

(b)

*

*

*

*

*

0.0 0.5 1.0 1.5

Concentration (C/C 0 )

HepG2 Cells 72 hr

(c)

*

*

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0 0.25 0.5 0.75 1

Concentration (C/C 0 )

24 hr

48 hr

72 hr

(d)

Fig 6 Measured absorbance in MTS assay on HepG2 cells at different time points: (a) 24 h, (b) 48 h, and (c) 72 h after the sludge extract was added (d) Cell viability (calculated from the MTS absorbance normalized by the corresponding control) in cell culture medium with different concentrations of sludge extract C 0 is the concentration

⁄

However, significant differences in the elemental composition of

these two bottom ashes can be observed for Fe element, which

the 20SWCG bottom ash contains higher concentration of Fe

ele-ment as compared to the PWG bottom ash This result can be

attributed to the high content of iron-compounds in the sewage

sludge

3.2.3 Mineralogical analysis of bottom ashes

X-ray diffraction was used to investigate the qualitative

miner-alogical characteristic of the bottom ashes arising from the

gasifi-cation of pure wood chips (PWG) and a mixture of 20 wt.%

sewage sludge and 80 wt.% wood chips (20SWCG) The XRD

pat-terns, as presented inFig 3, show that these two bottom ashes

have similar crystalline phases, indicating that both bottom ashes

contain similar chemical compounds The calcite (CaCO3) phase

are observed in both bottom ashes as a major component, whereas

the crystalline phases of ferrosilite ((Fe, Mg)SiO3), enstatite

(MgSiO3), quartz (SiO2), spurrite (Ca5(SiO4)2CO3) and maghemite

(c-Fe2O3) are also observed as the minor components This result

is consistent with that ofTafur-Marinos et al (2014)which also

reported the presence of those phases in the bottom residues from

the pelletized wood pyro-gasification However, amount of

iron-containing phases present in these two bottom ashes is slightly

dif-ferent The 20SWCG bottom ash contains higher amount of the

iron-containing phases i.e hematile (a-Fe2O3), metallic iron (Fe0)

as well as maghemite (c-Fe2O3) This is mainly due to the fact that

the sewage sludge obtained from wastewater treatment plant

con-tains high content of iron compounds

3.2.4 Organic compounds (e.g PAHs) present in the bottom ashes

Fig 4shows the GC–MS analysis of PAHs in the standard

solu-tion and bottom ash extracts All samples showed many peaks,

with some poorly resolved or overlapping ones Each peak in the chromatograms was matched with the NIST database to identify the components present, and compounds with P90% similarity were reported inTable 3 Using a series of diluted calibration stan-dards, the detection limit for the 16 priority PAHs was determined

to be 0.2–0.5 ppm Similarity search with the NIST database did not identify any of the samples to contain any of the 16 PAHs com-pounds in question Table 3shows the compounds found in the respective samples All samples consisted of a variety of organic compounds from different functional groups, such as alkanes, alkenes, carboxylic acid, ester and alcohol, together with one ben-zene derivative Aliphatic compounds found in all the samples are mainly long chain, with above 10 carbon atoms C9–C16 aliphatic fractions are considered to be much less toxic compared to aromat-ics Of smaller aromatic compounds, benzene is characterized as the most hazardous while its derivatives are of lower toxicity (WHO, 2004;U.S Environmental Protection Agency, 2010) Comparing the organic compounds found in both extracts of bottom ashes from PWG and 20SWCG, all are found to be relatively similar, with no additional PAH present in either sample In addi-tion, cytotoxicity of some PAHs has been determined in earlier studies For 48 h exposure, the PAH concentration resulting in 20% viability of HepG2 is 1.4 ppm for benzo[a]pyrene (Babich

et al 1988), 8.23 ppm Benzo[a]anthracene and 5.69 ppm for benzo[k]fluoranthene (Song et al 2011) This indicates that in our study, these PAHs of concentration close to the detection limits would not be significantly toxic Moreover, it is worthwhile to mention that the low solubility of PAHs in an aqueous environ-ment results in the low leaching of PAHs compounds (Park et al

2009), and thus it is reasonable to be expected that the toxicity properties of bottom ashes, which will be discussed in the follow-ing sections, are mostly caused by other factors

0

0.2

0.4

0.6

0.8

1

1.2

0.0 0.2 0.4 0.6 0.8 1.0

Concentration (C/C 0 )

24 hr

48 hr

72 hr

(a)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0 0.2 0.4 0.6 0.8 1.0

Concentration (C/C 0 )

24 hr

48 hr

72 hr

(b)

0 0.2 0.4 0.6 0.8

1 1.2 1.4

0.0 0.2 0.4 0.6 0.8 1.0

Concentration (C/C 0 )

24 hr

48 hr

72 hr

(c)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0.0 0.2 0.4 0.6 0.8 1.0

Concentration (C/C 0 )

24 hr

48 hr

72 hr

(d)

Fig 7 Viability of MRC-5 cells in different concentrations of extract of bottom ash from: (a) PWG and (b) 20SWCG after the extracts were neutralized by nitric acid; (c) and

3.3 Viability of MRC-5 and HepG2 cells in the sewage sludge extract

Fig 5shows the absorbance in the MTS assay of MRC-5 cells

after the cells were exposed to sludge extract solution for 24 h

The cells in contact with the sludge extract solution of

concentra-tion C0displayed significantly lower viability than those cultured

in medium at lower sludge extract concentrations When the

origi-nal solution was diluted by 10 times (corresponding to a

concen-tration C/C0 of 101), the cell viability greatly improved In

addition, for concentrations lower than 102, no differences were

observed in the absorbance between the wells with sludge extracts

and the control (P < 0.05)

Since changes in cell viability occurred mainly for C/C0> 102, the dilution factor was reduced in subsequent assays in an effort

to focus the investigation on toxicity in the most sensitive concen-tration range As shown inFig 6a, viability of HepG2 increased with decreasing concentrations of sludge extract, although the change was less significant when the C/C0was lower than 0.25 A comparison between Fig 6a and b shows that, with increasing exposure duration, the toxic effects became more significant even for low concentrations of sludge extract.Fig 6c shows the same trend, where the absorbance with C/C0= 0.05 (or in other words, after 20 times dilution of original sludge extract) was less than half

of the control reading As summarized inFig 6d, the original sludge

0.25

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Fig 8 Morphology of MRC-5 cells at different time points (24 h, 48 h and 72 h) after exposure to extracts of bottom ash from (a) PWG and (b) 20SWCG after the extracts were

extract induced the same toxicity effect regardless of exposure

duration between 24 h to 72 h; the medium with C/C0= 0.025

showed no significant toxicity below 72 h of exposure

3.4 Viability of MRC-5 and HepG2 cells in bottom ash extracts

Fig 7summarizes the cell viability of MRC-5 and HepG2 cells

after the cells were exposed to the extracts of bottom ash from

PWG and 20SWCG As shown inFigs 7a and b, the viability of

MRC-5 cells decreased sharply with increasing concentrations of

bottom ash extracts for both PWG and 20SWCG – almost all the

cells survive at C/C0= 0.05, but few remained alive after C/C0 is

increased to 0.25 For concentrations between 0.05 and 0.25, the

cell viability increased with time, possibly reflecting a recovery

of growth after the initial damage to cells For example, cell viabil-ity for C/C0= 0.1 was about 20% at 24 h, but increased to 35% and 82% for PWG and 20SWCG, respectively, at 72 h

Although HepG2 viability also decreased with increasing con-centration, it displayed different features, as shown in Figs 7c and d The bottom ash from PWG was relatively less toxic to HepG2 cells as compared to 20SWCG For example, for the same concen-tration of C/C0= 0.25, cell viability at 24 h was 70% for PWG but 10% for 20SWCG; and for C/C0= 0.5, the corresponding viability was 35% and 0%, respectively On the other hand, the HepG2 cell viability at 72 h was lower than at 24 h, indicating that damage

to cells was enhanced with increased duration of exposure

It is not surprising that the cytotoxic effects become more severe with increasing exposure time and concentration The

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0.025

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(b)

ference in viability between the different cell types exposed to the

same toxins is also expected However, cell viability is not the sole

parameter for evaluating the toxicity of bottom ash extract For

example, cells may be viable in certain environment, but they

may not be able to perform their normal biological functions, such

as growth, division and secretion Therefore, toxicity effects should

be simultaneously characterized using more parameters, including

cell morphology which is discussed in the following section

3.5 Cell morphology in bottom ash extracts

Besides cell viability, cell morphology can also reveal

informa-tion about toxic effects on human cells If the morphology of cells

appear different from their normal shape, it could indicate that they are not healthy, possibly resulting from a toxic environment which is unsuitable for healthy cell growth While cell morphology can be quantitatively evaluated using the ratio of major axis to minor axis, the use of LIVE/DEADÒ staining coupled can provide useful qualitative results as shown below

Fig 8shows the morphology of MRC-5 cells immuno-stained using the LIVE/DEADÒ Kits for mammalian cells after they were cultured in the extracts of bottom ash from PWG and 20SWCG for 24–72 h Live cells are stained green and dead cells display red fluorescence, respectively In a favorable environment, fibro-blastic cells are usually bipolar or multipolar and have elongated shapes (for example, at 72 h for C/C0= 0 inFigs 8a and b) A

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Fig 9 Morphology of HepG2 cells at different time points (24 h, 48 h and 72 h) after exposure to extracts of bottom ash from (a) PWG and (b) 20SWCG after the extracts was