w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3

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Comprehensive microbial analysis of combined

mesophilic anaerobicethermophilic aerobic
process treating high-strength food wastewater

Hyun Min Jang a, Jeong Hyub Ha a,b,*, Jong Moon Park a,b,c,**,
Mi-Sun Kim d, Sven G. Sommer e
a
School of Environmental Science and Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
b
Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang 790-
784, Republic of Korea
c
Division of Advanced Nuclear Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
d
Biomass and Waste Energy Laboratory, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon
305-343, Republic of Korea
e
Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University
of Southern Denmark, Niels Bohrs All
e 1, DK-5230 Odense M, Denmark

article info abstract

Article history:
Received 1 October 2014
Received in revised form
27 January 2015
Accepted 28 January 2015
Available online 7 February 2015
Keywords:
High-strength food wastewater
Combined biological process
Methane production
Pyrosequencing
Quantitative real-time PCR (qPCR)
A combined mesophilic anaerobicethermophilic aerobic process was used to treat high-
strength food wastewater in this study. During the experimental period, most of solid
residue from the mesophilic anaerobic reactor (R1) was separated by centrifugation and
introduced into the thermophilic aerobic reactor (R2) for further digestion. Then, ther-
mophilic aerobically-digested sludge was reintroduced into R1 to enhance reactor perfor-
mance. The combined process was operated with two different Runs: Run I with hydraulic
retention time (HRT) ¼ 40 d (corresponding OLR ¼ 3.5 kg COD/m3 d) and Run II with
HRT ¼ 20 d (corresponding OLR ¼ 7 kg COD/m3). For a comparison, a single-stage meso-
philic anaerobic reactor (R3) was operated concurrently with same OLRs and HRTs as the
combined process. During the overall digestion, all reactors showed high stability without
pH control. The combined process demonstrated significantly higher organic matter
removal efficiencies (over 90%) of TS, VS and COD and methane production than did R3.
Quantitative real-time PCR (qPCR) results indicated that higher populations of both bacteria
and archaea were maintained in R1 than in R3. Pyrosequencing analysis revealed relatively
high abundance of phylum Actinobacteria in both R1 and R2, and a predominance of phyla
Synergistetes and Firmicutes in R3 during Run II. Furthermore, R1 and R2 shared genera
(Prevotella, Aminobacterium, Geobacillus and Unclassified Actinobacteria), which suggests
synergy between mesophilic anaerobic digestion and thermophilic aerobic digestion. For

* Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 8315; fax: þ82 54 279 8659.
** Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 2275; fax: þ82 54 279 8659.
E-mail addresses: jeonghha@postech.ac.kr (J.H. Ha), jmpark@postech.ac.kr (J.M. Park).
http://dx.doi.org/10.1016/j.watres.2015.01.038
0043-1354/© 2015 Elsevier Ltd. All rights reserved.
292 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
archaea, in R1 methanogenic archaea shifted from genus Methanosaeta to Methanosarcina,
whereas genera Methanosaeta, Methanobacterium and Methanoculleus were predominant in
R3. The results demonstrated dynamics of key microbial populations that were highly
consistent with an enhanced reactor performance of the combined process.
1. Introduction
The amount of food waste (FW) significantly increases and it is
estimated about 1.3 billion tons in 2011 (Gustavsson et al.,
2011). In Korea alone, ~4 million tons of FW per year has
been generated and it accounts for over 26% of total municipal
solid waste (MSW) generation (MOE, 2012). Typically collected
FW is recycled as feedstuff, fertilizer and soil amendment
through washing and composting processes. Food wastewater
(FWW) is a byproduct of the recycling of food waste (FW).
Korea produces >3.5 million tons of FWW per year (MOE, 2012)
that contains a high concentration of organic matter. Tradi-
tional methods to treat FWW have adverse environmental
effects, such as contamination of drainage system and mal-
odor production, so development of appropriate management
methods has become an environmental and public concern
(Shin et al., 2001).
Effective degradation of the organic fraction in the FWW is
of key importance to reduce its disagreeable environmental
effects. Anaerobic digestion (AD) is considered as one of the
promising practical technology that exploits microbial meta-
bolic abilities to reduce the organic fraction in FWW by con-
verting the organics to methane (Li et al., 2011); AD can also
prevent fugitive emissions more than can other technologies
(Levis et al., 2010). Meanwhile, to increase the efficiency of AD,
two-stage or multi-stage AD processes (i.e., temperature-
phased anaerobic digestion (TPAD) or separated acid-
ogenicemethanogenic step) have been developed (Pervin
et al., 2013; Shin et al., 2010). Also, a combined anaerobic-
aerobic process has been used due to the potential to
remove organic matter and produce methane efficiently
under two extremely different redox conditions (Novak et al.,
2003; Tomei et al., 2011).
Microbial degradation of FWW is a synergistic and complex
process that involves a diverse assemblage of bacteria and
methanogenic archaea. Thus, to achieve efficient degradation
of FWW, the AD reactor should support a delicately-balanced
composition of bacteria and archaea to ensure that they
interact effectively for the process. Therefore, understanding
of the microbial communities in the AD and their responses to
environmental changes such as pH, temperature, substrate,
organic loading rate (OLR) and hydraulic retention time (HRT)
might provide valuable information that can be used to opti-
mize operation of AD.
Various molecular microbiology techniques have been
applied to understand the microbial communities in various
types of AD and target organic wastes. Shin et al. (2010) and
Regueiro et al. (2012) used denaturing gradient gel electro-
phoresis (DGGE), quantitative real-time PCR (qPCR) and
© 2015 Elsevier Ltd. All rights reserved.
fluorescence in situ hybridization (FISH) techniques to quan-
tify and qualify microbial community structures in lab or full-
scale anaerobic digesters. Hori et al. (2006) and Niu et al. (2013)
applied single-strand conformation polymorphism (SSCP) and
terminal restriction fragment length polymorphism (T-RFLP)
to identify the characteristic microorganisms during ther-
mophilic AD. However, the throughput and sensitivity of
these molecular techniques are not sufficient to cover the
overall taxonomic distribution; they are also time-consuming
and require highly-skilled operation (Ercolini, 2004; Gao and
Tao, 2012).
Recently-developed sequencing technologies provide
higher throughput of sequence data in complex biological
samples without the limitations of existing culture-
independent molecular techniques. One such recent tech-
nique is 454-pyrosequencing, which is based on sequencing
by synthesis and relies on detection of pyrophosphate
released during nucleotide incorporation (Margulies et al.,
2005). Pyrosequencing has been used to investigate microbial
community structure in 21 (Sundberg et al., 2013) and 7 (Lee
et al., 2012) full-scale AD treating various organic matters
and at different temperature ranges. Pervin et al. (2013) also
indicated the applicability of pyrosequencing in multi-stage
AD of sewage sludge.
In this study, we proposed a novel combined biological
process which consists of mesophilic anaerobic digestion
(MAD) combined with thermophilic aerobic digestion (TAD)
and solid separation unit for treating high-strength FWW. To
compare the organic matter removal efficiency and methane
production, a lab-scale combined process and conventional
single-stage AD were operated simultaneously with the same
feedstock for a period of 204 d. We also used pyrosequencing
and qPCR methods to investigate microbial community
structure and population in the proposed process. To the best
of our knowledge, this is the first report on the reactor per-
formance and phylogenetic diversity of bacteria and archaea
in combined MAD-TAD process during the treatment of high-
strength FWW.
2. Material and methods
2.1. Preparation of feedstock
The feedstock used in this study was collected from a FWW
storage tank in FW recycling facility in Pohang city, South
Korea. This facility treats ~180 ton/d of FW generated from
public and private sectors, and generates ~50 ton/d of FWW.
Samples from a FWW storage tank were filtered (1.0-mm
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3 293

During the experiment, most of the solids from R1 were

Table 1 e Characteristics of FWW used in this study.
Parameters
pH
Total Solids (TS) (g/L)
Volatile Solids (VS) (g/L)
VS/TS (%)
Total Chemical Oxygen
Demand (TCOD) (g/L)
Soluble COD (SCOD) (g/L)
Total nitrogen (TN) (g/L)
Soluble TN (STN) (g/L)
NHþ4 eN (g/L)
Total phosphorus (TP) (g/L)
Soluble TP (STP) (g/L)
Lactic acid (g COD/L)
Acetic acid (g COD/L)
Propionic acid (g COD/L)
Butyric acid (g COD/L)
Succinic acid (g COD/L)
Total VFA (g COD/L)a
Total Organic acids (g COD/L)b
a
Sum of acetic-, propionic- and butyric acid.
b
Sum of all organic acids.
separated by centrifugation (8000 rpm, 20 min) and recycled
Values (average ± standard into R2 for further digestion. Then, thermophilic aerobically-
deviation)
digested organic matter from R2 was reintroduced into R1 to
4.31 ± 0.02 enhance methane production by exploiting the synergy be-
118.49 ± 3.54 tween MAD and TAD. MAD and TAD were operated with
106.52 ± 3.41
working volumes of 11 and 1 L, respectively (Table S1). Addi-
90.5 ± 0.69
tionally, conventional single-stage MAD (R3; control process)
139.58 ± 2.79
was operated with working volume of 6L under the same total
90.46 ± 1.53 HRTs (40- and 20-d) as the combined process for 204 days to
1.94 ± 0.15 compare the reactor performance and the microbial commu-
1.02 ± 0.35 nities in the reactors. Although same total HRTs were applied,
0.57 ± 0.03 the HRTs in R1 (22- and 13.75-d) were shorter than those of R3,
2.22 ± 0.23
due to the recirculation from R2 to R1. More details about the
1.58 ± 0.02
40.74 ± 1.04
operating conditions are presented in Table S1. The seed for
14.35 ± 0.59 anaerobic reactors was obtained from a successfully-operated
3.32 ± 0.31 full-scale AD plant (Daegu, South Korea); the seed for the
9.94 ± 0.51 thermophilic aerobic reactor was obtained from an auto-
1.12 ± 0.04 thermal thermophilic aerobic digestion (ATAD) pilot plant
27.61 ± 1.02
(Daejeon, South Korea).
69.47 ± 1.46
2.3. DNA extraction and pyrosequencing

Total genomic DNA used for pyrosequencing and qPCR was

sieve) to remove inert materials (mainly eggshell, plastic and
vinyl), then distributed in 3-L bottles and stored at 25  C until
use. The characteristics of FWW were assayed (Table 1); the
main characteristics were as follows: pH 4.31 ± 0.02, total
solids (TS) 118.49 ± 3.54 g/L, volatile solids (VS) 106.52 ± 3.41 g/
L, total chemical oxygen demand (TCOD) 139.58 ± 2.79, soluble
COD (SCOD) 90.46 ± 1.53 g/L, total organic acid (TOA)
64.47 ± 1.46 g COD/L and total volatile fatty acid (TVFA)
25.80 ± 0.94 g acetic acid/L.
2.2. Reactor operation
The combined process (Fig. 1) for high-strength FWW treat-
ment consists of a MAD process (R1) with a TAD process (R2).
extracted from samples at steady state of each Run (102 and
204 days) as described previously (Jang et al., 2014). Hyper-
variable regions within bacterial and archaeal 16s rRNA genes
were amplified by PCR using universal primers (Table S2); for
bacteria: Bac27F (50 -adaptor A-Barcode-AC-GAG TTT GAT CMT
GGC TCA G-30 )/Bac541R (50 -adaptor B-Barcode-AC-WTT ACC
GCG GCT GCT GG-30 ); for archaea: Arc344F (5'-adaptor A-Bar-
code-GA-YGG GGY GCA SCA GGS G-30 )/Arc927R (50 -adaptor B-
Barcode-GA-CCC GCC AAT TCC TTT AAG TTT C-30 ) (Jung et al.,
2013).
PCR amplification was conducted using FastStart High Fi-
delity PCR system (Roche, Branford, CT). The protocol was
composed of: (1) initial denaturation at 94  C for 4 min; (2) 35
cycles of 94  C for 15 s, 55  C for 45 s, and 72  C for 1 min; (3) a
final extension at 72  C for 8 min. The samples were purified

Fig. 1 e Conceptual schematic diagram of (a) combined and (b) control process (pump flow rates Q1e7 are described in
Supplementary material Table S1).
294 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
using PCR purification kit (Solgent, Korea), then DNA con-
centrations were measured using a fluorometer with Quant-
iT™ PicoGreen® dsDNA Assay Kit (Invitrogen ™, California).
Equal amounts of PCR amplicons from each sample were
pooled, then pyrosequencing was performed with 454-GS-FLX
Titanium (Roche, Branford) using the massively parallel
pyrosequencing protocol by a sequencing provider (Macrogen,
Korea).
2.4. Analysis of pyrosequencing data
Pyrosequencing raw data were processed and analysed using
the Ribosomal Database Project (RDP) Pyrosequencing Pipeline
Initial Process (http://pyro.cme.msu.edu/) (Cole et al., 2009).
The sequencing reads were assigned to specific samples based
on their unique barcodes, and then the barcodes and primers
were trimmed. Sequencing reads with lengths <400 bp, an
average quality score <20, or >2 ambiguous bases (N) were
removed. Chimeric sequences were identified and removed
using the MOTHUR program (Schloss et al., 2009). The multiple
clean sequences were aligned using the fast, secondary-
structure aware INFERNAL aligner (Nawrocki and Eddy,
2007). The aligned sequences were clustered using complete
linkage-clustering method into operational taxonomic units
(OTUs) defined by 97% similarity. The Shannon-Weaver index
(Shannon and Weaver, 1963), Chao1 richness indices (Chao
and Bunge, 2002) and evenness were calculated using clus-
tered sequence data by the Shannon Index and Chao1 esti-
mator of the RDP pyrosequencing pipeline. Rarefaction curves
(Colwell and Coddington, 1994) and Good's coverage
(G ¼ 1  (n/N), where n is the number of singleton phylotypes
and N is the total number of sequences in the sample) were
also constructed with a 3% dissimilarity cut-off value using
the RDP pyrosequencing pipeline. The taxonomic assignment
of clean sequences obtained from each sample was conducted
using the SILVA ribosomal RNA gene database (Quast et al.,
2013). Principal coordinate analysis (PCoA) of bacterial and
archaeal were performed to confirm the multiple commu-
nities from the UniFrac analysis.
2.5. qPCR analysis
To investigate the bacterial (all reactors) and archaeal (R1
and R3) population at the two different HRTs applied, qPCR
amplification and fluorescence detection were performed
using an Applied Biosystems 7300 qPCR system (Applied
Biosystems, Forster City, USA). Two primer sets were used
for quantification of 16S rRNA gene copies; for bacteria:
Bac338F (50 -ACT CCT ACG GGA GGC AG-30 )/Bac805R (50 -GAC
TAC CAG GGT ATC TAA TCC-30 ), for archaea: Arc787F (50 -ATT
AGA TAC CCS BGT AGT CC-30 )/Arc1059R (50 -GCC ATG CAC
CWC CTC T-30 ) (Jang et al., 2014). Ten-fold serial diluted (101
to 109 copies) 16S rRNA genes from representative strains (for
bacteria: Escherichia coli K12 (DSM 1607); for archaea: Meth-
anomicrobium mobile BP (DSM 1539)) was used to construct the
standard curve for each primer set. qPCR and calculation of
16S rRNA gene copies of target groups in each sample were
conducted as described previously (Lee et al., 2008; Whelan
et al., 2003).
2.6. Physico-chemical analytical methods
Standard Methods (APHA-AWWA-WEF, 1998) were used to
characterize physico-chemical properties (TS, VS, COD, total
nitrogen (TN), total alkalinity (TA) and total phosphorus (TP))
of samples. After centrifugation (5000 rpm, 30 min) and
filtering (0.45-mm pore size), the filtrate was analysed for sol-
uble COD (SCOD), soluble TN (STN), and soluble TP (STP). The
concentrations of cations and anions were determined by ion
chromatography (ICS-1100 and 1000, DIONEX Co., USA). The
pH and oxidation/reduction potential (ORP) in the reactors
were continuously measured using a pH meter (405-DPAS-SC-
K85, METTLER TOLLEDO, Switzerland) and an ORP meter (Pt-
4805, METTLER TOLLEDO, Switzerland). Organic acids
including VFAs were quantified using a high-performance
liquid chromatography (HPLC, Agilent Technology 1100 se-
ries, Agilent Inc., USA) equipped with a column (Aminex HPX-
87H, BIORAD Inc., USA), refractive index detector (RID), and
diode array detector (DAD). The total volume of biogas pro-
duced from the reactor was quantified using the water
displacement method; biogas composition was determined
using a gas chromatography (Model 6890N, Agilent Inc., USA)
equipped with a pulsed discharge detector (PDD). The quan-
tified organic acid values were converted theoretically to g
COD/L and g acetic acid/L. TOA and TVFA were calculated as
the sum of the organic acids and of the VFAs (acetic-, propi-
onic- and butyric acid), respectively.
3. Results and discussion
3.1. Reactor stability and performance
Samples from the reactor were taken at intervals of 3 days to
measure the change of physico-chemical characteristic of
reactor. In this study, state of reactor was considered as a
steady state when reactor was operated without significant
change of physico-chemical characteristic and performance
over two-month (Run I ¼ ~102 d, Run II ¼ ~204 d). All reactors
showed high stability throughout digestion without pH con-
trol (Table 2), even though FWW used in this study had high
concentrations of organic matter and organic acids, and low
pH (~4.3) (Table 1). This stability was not expected, because
high-strength and acidic feedstock are major environmental
stresses during the MSW treatment (Salminen and Rintala,
2002). Both anaerobic reactors (R1 and R3) were maintained
in the range of 6.71  pH  to 7.54, 2.20  TA  to 3.31 g
CaCO3/L, and 397  ORP  413 mV. In R2, relatively high
pH (~8.25) and low TA (~1.45 g CaCO3/L) were maintained
throughout digestion, mainly due to continuous aeration
which causes ammonia and CO2 stripping under thermo-
philic aerobic conditions (Juteau et al., 2004a). In R2, feeding
caused ORP to drop initially to 38 mV, but within 3 h after
feeding, ORP had recovered to 67 mV and maintained aerobic
condition as in recent reports (Cho et al., 2013; Jang et al.,
2014). According to these results, substantial microbial
communities were established in all reactors, particularly in
R1 and R2 under extremely different temperature and redox
conditions.
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3 295

Table 2 e Summary of the reactor performance of combined (R1 and R2) and control (R3) processes at the two different
conditions.
Parameters Run I (HRT: 40 d) Run II (HRT: 20d)
Combined process R3 (control) Combined process R3 (control)
R1 (MAD) R2 (TAD) Final effluent R1 (MAD) R2 (TAD) Final effluent
pH 7.43 ± 0.11 8.25 ± 0.04 7.43 ± 0.11 7.33 ± 0.09 7.37 ± 0.01 8.21 ± 0.02 7.37 ± 0.01 6.75 ± 0.04
TA (g CaCO3/L) 3.22 ± 0.07 1.45 ± 0.08 3.22 ± 0.07 3.29 ± 0.02 3.22 ± 0.02 1.43 ± 0.02 3.22 ± 0.02 2.23 ± 0.03
ORP(mV) 405 ±8 38a ~ 67b e 402 ± 4 403 ± 6 37a ~ 67b e 402 ±3
TS (g/L) 40.06 ± 1.99 56.68 ± 0.49 8.88 ± 0.21 37.74 ± 0.28 51.08 ± 0.82 72.12 ± 0.28 8.89 ± 0.19 43.68 ± 0.12
VS (g/L) 20.47 ± 1.01 29.80 ± 0.26 3.94 ± 0.18 20.63 ± 0.31 26.10 ± 0.42 37.92 ± 0.15 3.98 ± 0.22 23.88 ± 0.24
TCOD (g/L) 30.92 ± 0.92 47.29 ± 0.26 9.49 ± 0.58 29.98 ± 0.58 43.49 ± 0.39 61.42 ± 0.42 9.07 ± 0.06 40.32 ± 0.42
SCOD (g/L) 3.43 ± 0.03 3.29 ± 0.11 3.43 ± 0.03 2.11 ± 0.08 4.81 ± 0.11 4.06 ± 0.04 4.81 ± 0.11 3.20 ± 0.01
TOA (g COD/L) 2.86 ± 0.07 e 2.86 ± 0.07 0.89 ± 0.01 3.28 ± 0.01 e 3.28 ± 0.01 1.99 ± 0.05
Acetic acid (g COD/L) 1.52 ± 0.01 e 1.52 ± 0.01 0.89 ± 0.01 1.73 ± 0.01 e 1.73 ± 0.01 1.24 ± 0.03
Propionic acid (g COD/L) 1.43 ± 0.03 e 1.43 ± 0.03 e 1.55 ± 0.01 e 1.55 ± 0.01 0.75 ± 0.01
TVFA (g acetic acid/L) 2.67 ± 0.05 e 2.67 ± 0.05 0.83 ± 0.01 3.07 ± 0.01 e 3.07 ± 0.01 1.86 ± 0.05
TVFA/TA (g acetic acid/g 0.83 ± 0.09 e 0.83 ± 0.09 0.25 ± 0.02 0.95 ± 0.01 e 0.95 ± 0.01 0.83 ± 0.06
CaCO3)
TS removal efficiency 92.52 ± 0.35 68.15 ± 0.75 92.54 ± 0.42 63.13 ± 0.39
(% of feed)
VS removal efficiency 96.31 ± 0.41 80.63 ± 0.48 96.30 ± 0.28 77.58 ± 0.27
(% of feed)
COD removal efficiency 93.20 ± 0.28 78.52 ± 0.24 93.51 ± 0.11 71.11 ± 0.65
(% of feed)
Biogas production rate 1.54 ± 0.03 1.00 ± 0.02 2.67 ± 0.04 2.00 ± 0.01
(L/L/d)
Methane content (%) 68.40 ± 0.31 63.10 ± 0.58 66.20 ± 0.48 59.42 ± 0.44
Methane production rate 1.05 ± 0.01 0.63 ± 0.01 1.77 ± 0.02 1.19 ± 0.01
(L CH4/L/d)
Methane yield (m3 CH4/kg 0.32 ± 0.01 0.23 ± 0.01 0.32 ± 0.01 0.24 ± 0.01
COD removed)
a
After the feeding.
b
About 3 h after the feeding.

A considerable amount of organic matter was degraded in
both combined and control processes. In Run I at steady state,
the control process achieved removal (%) of 68.15 ± 0.75 for TS,
80.63 ± 0.48 for VS and 78.52 ± 0.24 for COD; the combined
process achieved 92.52 ± 0.35 for TS, 96.31 ± 0.41 for VS and
93.20 ± 0.28 for COD (Table 2). In Run II at steady state, the
increase in OLR from 3.5 to 7 kg COD/m3 d caused higher
concentrations of TS, VS and COD in all reactors. After the
initial period of Run II, the control process showed slightly
decreased removal (%) (63.13 ± 0.39 for TS, 77.58 ± 0.27 for VS
and 71.11 ± 0.65 for COD) compared to Run I at steady state.
But the removal (%) in the combined process (92.54 ± 0.42 for
TS, 96.30 ± 0.28 for VS and 93.51 ± 0.11% for COD) in Run II at
steady state did not differ from those in Run I at steady state.
These results indicate that efficient degradation of separated
(concentrated) anaerobic sludge in R2 significantly increased
the removal efficiency in terms of TS, VS and COD. In addition,
the collaboration between R1 and R2 might provide remark-
able differences in organic removal between combined and
control processes (Miah et al., 2005).
To better understand the reactor performance, organic
acids that are known to be major intermediates were ana-
lysed. Only two organic acids (acetic and propionic acid) were
detected in both R1 and R3; no organic acids were detected in
R2 (Table 2). In Run I at steady state, TOA concentration was
2.86 ± 0.07 g COD/L in R1 (final effluent of combined process)
and 0.89 ± 0.01 g COD/L in R3. With increased OLR, increased
TOA concentration was observed in R1 (3.28 ± 0.01 g COD/L)
and R3 (1.99 ± 0.05 g COD/L). This suggests that HRT of R1 and
R2 were insufficient to allow consumption of all organic acids
in feedstock and produced in the reactors. The TOA concen-
tration was higher in R1 than R3, possibly because HRT was
shorter in R1 than in R3 during the overall digestion (Table S1).
Degradable organic matter recirculated from R2 might also
contribute to the relatively high concentration of TOA in R1.
Based on the organic acid analysis in the reactor, the TVFA/
TA ratio was calculated; this ratio has been used as an
acidification-risk parameter in AD (Kafle and Kim, 2013;
Schievano et al., 2012). In general, TVFA/TA < 0.4 is consid-
ered to be favourable for stable methanogenesis without in-
hibition by acidification (Rinco
n et al., 2008). In this study,
TVFA/TA was >0.8 in R1 in both Runs at steady state, and in R3
during Run II at steady state. This high TVFA/TA ratio is
mainly due to: (1) high concentration of easily-biodegradable
components (i.e., VS and SCOD) and >69 g COD/L of organic
acids in the feedstock (Table 1), and (2) relatively insufficient
retention time (especially R1). Although R1 and R3 showed
high TVFA/TA ratio, both R1 and R3 operated stably
throughout digestion. This observation is consistent with a
recent report that showed stable reactor operation with TVFA/
TA > 0.9 during the co-digestion of apple waste and swine
manure (Kafle and Kim, 2013). Further studies need to be
Geotechnical Engineering Bored pile design in stiff clay I: codes of
Volume 165 Issue GE4 practice
Vardanega, Kolody, Pennington, Morrison and
Simpson

Qwork: kN
100 200 300 400 500 600 700 800
10
11
12
13
Pile length, L: m

14
15
16
17
18
19
20
Conventional design AS2159-2009
UK EC7 (DA1-2) Irish EC7 (DA2)
Netherlands EC7 (DA3) SNiP
AASHTO

Figure 10. Unfactored working load plotted against pile length

(0.45 m diameter pile)

Qwork: kN
200 400 600 800 1000 1200 1400 1600 1800 2000
10
11
12
13
Pile length, L: m

14
15
16
17
18
19
20

Conventional design AS2159-2009

UK EC7 (DA1-2) Irish EC7 (DA2)
Netherlands EC7 (DA3) SNiP
AASHTO

Figure 11. Unfactored working load plotted against pile length

(0.9 m diameter pile)

15. Discussion and conclusion
The following observations are made based on the study
described in the current paper.
(a) The UK (DA1), Netherlands (DA3) and AS2159 calculations
give closely similar results (for this design example, using the
Æ-method of calculation) with a global FOS of just under 2.5.
The Irish DA2 approach gives a slightly higher FOS value.
The difference occurs when AASHTO and SNiP are
considered. AASHTO is a very conservative code as the
factors and the loading and resistance are very high.
AASHTO would be even more conservative if the design line

4·0
3·5
Global FOS
3·0
2·5
2·0
1·5
10 11 12 13 14 15 16
Pile length, L: m
Figure 12. Global FOS plotted against pile length (0.9 m
diameter pile)
(Equation 8a) was used instead of Equation 8c. The fact that
AASHTO is mainly a bridge code could be why the variable
loading factor of 1.75 is very high and why designs are very
conservative. The SNiP calculations are significantly less
conservative.
(b) Most codes have the flexibility of applying different factors to
the shaft and base resistance. The base is generally factored
higher as more uncertainty exists in the determination of what
the pile is founded in and how much the base is disturbed by
construction. The Australian and Russian codes use a single
reduction factor applied to the combined resistances.
(c) The Australian code is unique in that the engineer has input
into the factor of safety chosen by means of a simple risk
analysis approach. This recognises that site conditions dictate
the amount of uncertainty in the design to a certain extent
and that a ‘one-size-fits-all’ approach can constrain
engineering judgement which is crucial for good design. This
could also be seen in the Eurocode context as embodied in
the use of a ‘cautious estimate’, which is perhaps a more
abstract concept that achieves a similar result.
(d ) Direct comparison of the allowable working pile resistance,
Qwork , for each code is obscured by the fact that different
estimates of shear strength were used, especially in AASHTO
and SNiP where triaxial data only and liquidity index
correlations respectively are used to derive a cu profile and fi
profile respectively.
(e) There is little guidance in any of the design codes on how to
construct a design line for the shear strength profile. Some
codes specify (or imply) the use of average soil parameters
while Eurocode 7 (design by calculation) requires the use of
a ‘characteristic design line’ which is a ‘cautious estimate’.
Code drafters could adopt a statistical approach (e.g. mean or
5th percentile); however, it is considered that this ignores the
practice
Vardanega, Kolody, Pennington, Morrison and
Simpson
Conventional design
AS2159-2009
UK EC7 (DA1-2)
Irish EC7 (DA2)
Netherlands EC7 (DA3)
SNiP
AASHTO
17 18 19 20
causes of ground variability. The use of a ‘cautious estimate’
or similar concept does allow the engineer a degree of
flexibility in this respect. If the engineer accepts each data
point as equally valid then a design line could be derived
statistically. It does seem curious that partial factors can be
assigned without knowledge of how conservatively engineers
treat their soil data. If average soil values are to be used in
design then higher partial factors are needed than if 5th
percentile values are used. This is investigated further in the
companion paper (Vardanega et al., 2012).
( f ) A complication when comparing different codes of practice is
that permanent and variable loads are factored differently
from code to code. For a fair comparison of codes, the factors
on loads (actions) and resistances need to be brought
together. The key to success is that there is a clear
understanding between structural and geotechnical engineers
as to who applies the partial factors on actions.
(g) For bored piles in London Clay ‘Æ’ of 0.5 is generally
recommended. The AASHTO approach and the SNiP
approach use similar values of shaft resistance. AASHTO has
‘Æ’ of 0.55 dropping gradually as estimates of cu increase. In
other words this code penalises a high cu value.
(h) In SNiP the factor ªcf can be interpreted as similar to the ‘Æ’
concept as it reduces the shear strength of the clay around a
bored pile and relates to the method of installation. The use
of liquidity index (IL ) is not without basis as relationships
between IL and cu have been discussed (e.g. Muir Wood,
1983). Possibly, the use of IL in SNiP ‘works’ because it
indirectly measures values of cu , which relate to shaft friction.
(i) An interesting feature of the AASHTO approach is that the
SPT is not favoured for design; triaxial data are favoured.
This is despite SPT data sometimes displaying less scatter
than triaxial data (see Figures 4 and 5 and LDSA (2000)).
229
Geotechnical Engineering Bored pile design in stiff clay I: codes of
Volume 165 Issue GE4 practice
Vardanega, Kolody, Pennington, Morrison and
Simpson

( j) The major reason SNiP appears unconservative is that the

d ¼ 35

107.7
388.4
447.5
174.0
partial factor on resistance (1.4) and the partial factor on
actions (1.2) are both relatively low. It is not known if the
estimates of skin friction are conservative or not as the source
of the data in SNiP Tables 2 and 7 (Figures 11 and 12 in this

d ¼ 30

89.2
341.1
405.1
160.5
paper) is unclear. A comparison with Æcu values derived
suggests that they are high at shallow depth and low at
greater depth. Overall for the 12 m pile, there is little
difference between the SNiP representative resistance and

d ¼ 25

79.1
303.5
365.0
146.8
that derived from the ‘Æ’ method. It would be interesting to
know performance statistics for piled foundation systems
constructed under the SNiP framework.

d ¼ 20

74.1
275.6
329.2
133.6
Acknowledgements
Thanks must go to the Cambridge Commonwealth Trust and Ove
Arup and Partners for providing financial support to the first
author. The first author offers thanks to Professor Malcolm

d ¼ 15

61.4
236.0
288.5
119.8
Bolton for his supervision. The authors would also like to thank
Professor Harry Poulos for providing a draft copy of AS2159-
2009 for examination and for his advice on an early draft of the
paper.
d ¼ 10

52.2
203.7
252.2
106.6
Appendix – SNiP design charts
For the shaft resistance of piles in clay, cubic equations of the
form in Equation 42 were fitted to the data tables from SNiP. The
47.1
186.7
234.4
100.7
d¼8

regression coefficients are shown in Table 12 and the plotted

functions in Figure 13.

42: f i ¼ a(I L )3 þ b(I L )2 þ c(I L ) þ z

41.2
168.2
214.7
93.7
d¼6

For the base resistance of piles in clay, linear equations of the

form shown below were fitted to the data tables from SNiP. The
58.9
198.2
226.4
93.0
d¼5

regression coefficients are shown in Table 13 and the plotted

functions in Figure 14.
76.6
225.8
235.1
91.1
d¼4

43: R ¼ A(I L ) þ K
48.0
168.5
195.9
80.4

Depth of stratum: m
d¼3
displacement and bored piles, fi : kPa

200
1 2
180
3 4
Shaft resistance for driven,

Table 12. Fitted coefficients (Table 2, SNiP)

160 5 6
43.8
151.3
174.5
70.6
d¼2

140 8 10
120 15 20
100 25 30
35
80
90.1
230.7
206.0
67.2
d¼1

60
40
20
Stratum depth,

0
0 0·1 0·2 0·3 0·4 0·5 0·6 0·7 0·8 0·9 1·0 1·1
Liquidity index, IL
d: m

Figure 13. Graphical representation of Table 2 in SNiP 2.02.03-85

b
a

c
z
Geotechnical Engineering Bored pile design in stiff clay I: codes of
Volume 165 Issue GE4 practice
Vardanega, Kolody, Pennington, Morrison and
Simpson

Design resistance, R: d¼3 d¼5 d¼7 d ¼ 10 d ¼ 12 d ¼ 15 d ¼ 18 d ¼ 20 d ¼ 30 d ¼ 40

kPa below the pile tip

A 1054 1107 1196 1250 1446 1679 1911 2107 3300 5000
K 845 875 1116 1325 1541 1811 2088 2304 3300 4500

Table 13. Fitted coefficients (Table 7, SNiP)

Pile base depth: m

Balkema, Rotterdam, the Netherlands, pp. 301–309.
5000 3 5
10 NEN (Netherlands Standardization Institute) (2007) Eurocode 7:
4500 7
Base resistance at pile toe for

12 15 National Annex to NEN-EN 1997-1 – Geotechnical design –

4000 18 20 Part 1: General rules. NEN, Delft, the Netherlands.
bored piles, R: kPa

3500 30
3000
2500
2000
1500
1000
500
0 Poulos HG (2004) An approach for assessing geotechnical
0 0·1 0·2 0·3 0·4
Liquidity index, IL
Figure 14. Graphical representation of Table 7 in SNiP 2.2.03-85
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Transportation Officials) (2007) LRFD Bridge Design
Specifications, 4th edn. AASHTO, Washington, DC, USA
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Bond AJ and Simpson B (2010) Pile design to Eurocode 7 and the
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BSI (1986) BS 8004: 1986: Code of practice for foundations. BSI,
Milton Keynes, UK.
BSI (2007) National Annex to Eurocode 7: BS EN 1997-1-2004:
Geotechnical design – part 1: general rules. BSI, Milton
Keynes, UK.
BSI (2010) Eurocode 7: BS EN 1997-1-2004: Geotechincal design
– part 1: general rules. BSI, Milton Keynes, UK
(incorporating corrigenda February 2009).
LDSA (London District Surveyors Association) (ed.) (2000)
Guidance Notes for the Design of Straight Shafted Bored
Piles in London Clay. LDSA Publications, Bromley, UK,
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Meyerhof GG (1976) Bearing capacity and settlement of pile
foundations (11th Terzaghi Lecture). Journal of the
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NSAI (National Standards Authority Ireland) (2005) National
Annex Eurocode 7: I. S. EN 1997-1-2005: Geotechnical
design – Part 1: General rules. NSAI, Dublin, Ireland.
Patel D (1992) Interpretation of results of pile tests in London
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296 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
conducted to clarify the relationship between TVFA/TA ratio
and reactor performance during AD. Furthermore, alkalinity
addition may be required to alleviate the possible acidification
and to maintain appropriate pH condition for methanogenesis
when the proposed combined process is operated with OLR
>7.5 kg COD/m3 d.
The biogas production and methane content in both
combined and control processes were monitored throughout
digestion (Table 2). In Run I at steady state, the combined
process achieved 1.54 ± 0.03 L/L/d biogas production rate with
68.40 ± 0.31% methane content and the control process ach-
ieved 1.00 ± 0.02 L/L/d biogas production rate with
63.10 ± 0.58% methane content. In Run II at steady state, the
combined process achieved 2.67 ± 0.04 L/L/d biogas produc-
tion rate with 66.20 ± 0.48% methane content and the control
process achieved 2.00 ± 0.01 L/L/d biogas production rate with
59.42 ± 0.44%; in both cases, the biogas production was
higher, but the methane content lower, than in Run I at
steady state.
Methane production rate (MPR) and methane yield (MY)
were calculated from the data. The combined process ach-
ieved MPR ¼ 1.05 ± 0.01 (Run I at steady state) and 1.77 ± 0.02 L/
L/d (Run II at steady state) with stable MY ¼ 0.32 ± 0.01 m3 CH4/
kg CODremoved; these values were ~1.5 times higher than those
of the control process (MPR ¼ 0.63 ± 0.01 (Run I at steady state)
and 1.19 ± 0.01 L/L/d (Run II at steady state), MY ¼ 0.22e0.25 m3
CH4/kg CODremoved). These findings indicate that combined
process have potential advantages in terms of methane re-
covery as compared to control process. Therefore, it can be
inferred that the additional digestion in R2 of highly-
concentrated solid residue from R1 resulted in remarkable
degradation of organic matter and increased methane
production.
3.2. Quantification of total bacteria and archaea
Typically, microbial population (expressed as 16S rRNA gene
concentration) in biological reactor is strongly related to the
respond to environmental stress (Klappenbach et al., 2000).
Fig. 2 e Changes in 16S rRNA concentration of total bacteria, total archaea and ratio of archaea to total microbes (the sum of
bacteria and archaea) in different Runs I and II.
Therefore, measurement of 16S rRNA gene concentration is
useful during treatment of high-strength wastewater. qPCR
method was used to measure the concentration of 16S rRNA
gene copies of total bacteria and archaea (Fig. 2). In addition, it
is noteworthy to mention that PCR amplicons was not detec-
ted in R2 with archaea specific primer. This implies that
archaea within the solid residue from R1 completely lost their
activity and were rapidly degraded due to high microbial ac-
tivity under thermophilic aerobic conditions.
In spite of the relatively short HRT (5 d), bacterial 16s rRNA
gene concentration in R2 increased from 8.24 (±0.09)  1010 to
1.02 (±0.07)  1012 copies/mL (~12-fold increase) in Run I and
maintained higher population (~1012 copies/mL) throughout
digestion than in both R1 and R3. This difference in bacterial
populations might be due to the different growth character-
istics between aerobic and anaerobic bacteria. This result
corresponds to previous findings that stable and high bacterial
populations were maintained under thermophilic aerobic
conditions (Jang et al., 2014, 2013). Interestingly, R1 and R3
showed similar variation patterns of bacterial population, but
R1 maintained higher bacteria population
(2.21 (±0.07)  1011 copies/mL in Run I and
6.25 (±0.15)  1011 copies/mL in Run II) than did R3
(6.29 (±0.14)  1010 copies/mL in Run I and
2.15 (±0.11)  1011 copies/mL in Run II) throughout the
experiment (Fig. 2).
Similar to bacteria, archaea population in R1 and R3
increased as OLR increased and R1 showed higher archaea
population (2.79 (±0.25)  1010 copies/mL in Run I and
7.69 (±0.33)  1010 copies/mL at Run II) than did R3
(6.90 (±0.06)  109 copies/mL in Run I and
2.35 (±0.07)  1010 copies/mL Run II) throughout digestion.
Additionally, the percentage of archaea to total microbes was
slightly higher in R1 (11 ± 0.58%) than in R3 (9.8 ± 0.12%).
Collectively, the qPCR results demonstrated that the com-
bined process provided more suitable environment for growth
of both bacteria and archaea, thereby resulting in higher MPR
and methane yield as well as organic removal than did the
control process (Table 2).
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
Fig. 4 e Distribution and relative abundance of taxa in archaeal samples based on sequences derived from pyrosequencing.
Inner circle: composition at phylum level; outer circle: composition at order level. Sequences showing with percentage of
reads >1.0% in all samples were grouped into ‘Others’.
the higher concentration of 16S rRNA copies in both R1 and R2
than that of R3 during the overall digestion (Fig. 2). Thus, the
results from this study confirmed the hypothesis that R1 and
R2 worked synergistically, but further investigation and
characterization of uncultured and unclassified bacteria will
be required to clarify their metabolic functions and activities
in the proposed combined process.
3.5. Changes in methanogen species
During typical AD, complex organic matter can be degraded
and fermented by bacteria and converted finally into
methane, by methanogens as part of their energy metabolism
(Garcia et al., 2000). Understanding the taxonomic distribution
and properties of methanogens allows us to infer methano-
genic pathway in the reactor. 16S rRNA gene sequences ob-
tained from pyrosequencing of archaeal samples were
assigned to phylum Euryarchaota, which includes metha-
nogens (Fig. 4). Furthermore, >99.5% of sequences were
assigned to classified methanogen orders. This means that
most of archaea found in this study were involved in meth-
anogenesis. All archaeal samples showed lower diversity and
taxonomic distribution than those of bacterial samples;
particularly, the Shannon-Weaver Index in R1 showed the
lowest value (0.44) in Run II (Table S3).
The taxonomic distributions of the archaeal communities
at the order level differed distinctly between the anaerobic
299
reactors (Fig. 4). Similar to this finding, PCoA plot of archaeal
samples also indicated distinct regional separation (Fig. S2b).
In R1, the relative abundance of the aceticlastic methanogen
of order Methanosarcinales increased from 79.29% (Anaerobic
seed) to 92.43% in Run I and to 95.26% in Run II. Meanwhile, in
R3, this order decreased to 62.07% in Run I and 55.51% in Run
II, whereas the proportion of hydrogenotrophic methanogens
increased from 20.57% (Anaerobic seed) to 37.65% (order
Methanobacteriales, 17.98%; order Methanomicrobiales, 19.67%)
in Run I and to 44.13% (order Methanobacteriales, 16.94%; order
Methanomicrobiales, 27.19%) in Run II. This significant increase
of the hydrogenotrophic methanogens strongly indicates that
the hydrogenotrophic methanogenesis pathway might
contribute to methane production in R3, but that is not a
predominant pathway in R1. Additionally, the high abun-
dance of hydrogenotrophic methanogens in R3 was supported
by the large proportions of syntrophic bacteria (i.e., genera
Anaerobaculum and Clostridium) (Fig. 5a), which can produce
H2, an important electron source for hydrogenotrophic
methanogenesis (Wirth et al., 2012).
The archaea sequences were also classified at the genus
level (Fig. 5b). In anaerobic seed, the sequences were sub-
divided into 19 genera, but six of these genera (Unclassified
Methanobacteriales, Methanosphaera, Methanobrevibacter, Meth-
anolinea, Unclassified Methanomicrobiales incertae sedis and
Unclassified Archaea) were never detected in R1 or R3. During
Run I, Methanosaeta (42.93%) and Methanosarcina (44.54%)
300 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3

Fig. 5 e Heat map (constructed using R software with gplots package) of (a) top 25 bacterial genera and (b) top 20 archaeal
genera. The colour intensity of scale indicates relative abundance of each genus. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
(belong to order Methanosarcinales) were the two main genera
in R1. Interestingly, during Run II, the relative abundance of
genus Methanosarcina reached up to 95.02%, whereas members
of the genus Methanosaeta were completely inhibited or
washed out in R1. In contrast, in R3, >50% of sequences was
assigned to genus Methanosaeta, whereas members of the
genus Methanosarcina were never detected. Also, Meth-
anobacterium (belong to order Methanobacteriales; 17.58% in Run
I and 16.64% in Run II) and Methanoculleus (belong to order
Methanomicrobiales; 17.11% in Run I and 24.64% in Run II) were
the two main genera in R3. These genera are related to
hydrogenotrophic methanogenesis and are predominant
during AD of food-related wastes (Kim et al., 2014).
Typically, members of genus Methanosarcina can produce
methane by both aceticlastic and hydrogenotrophic meth-
anogenesis pathways (Garcia et al., 2000). Furthermore, in AD
the predominant methanogenic genera change from Meth-
anosaeta to Methanosarcina, as a result of changes in operating
parameters, particularly by increase in VFAs (especially acetic
acid) and decrease in HRT (De Vrieze et al., 2012; McMahon
et al., 2004). The change occurs mainly because these genera
have different growth characteristics. Members of genus
Methanosarcina can achieve stable growth at higher concen-
tration of VFAs and have higher specific growth rates than
genus Methanosaeta (Conklin et al., 2006). Thus, the relatively
high concentration of TVFA (3.07 ± 0.01 g acetic acid/L) (Table
2) and short HRT (13.75 day) (Table S1) during Run II probably
contributed to the predominance of genus Methanosarcina in
R1. Given this information, the predominance of Meth-
anosarcina in the methanogenic community in R1 strongly
indicates that members of the genus probably have important
functions in methanogenesis and may lead to a better per-
formance with stable conditions at low HRT, compared to R3.
4. Conclusions
In this study, a combined MAD (R1)-TAD (R2) process was used
to treat high-strength food wastewater. The specific conclu-
sions can be written as follows:
 The proposed combined MAD (R1)-TAD (R2) process
showed efficient treatment of organic matter removal (i.e.,
COD, TS and VS), and higher methane production than did
the control process (R3).
 Higher populations (based on the concentration of 16s
rRNA gene copy number) of both bacteria and archaea were
maintained in R1 than in R3 during the entire digestion.
 R1 and R2 shared a significant proportion of bacterial
genera (Prevotella, Aminobacterium, Geobacillus and Unclas-
sified Actinobacteria) and the predominant genus of meth-
anogenic archaea shifted from Methanosaeta to
Methanosarcina in R1.
 The qPCR and high-throughput pyrosequencing results
demonstrated that the synergisms between MAD and TAD
improved reactor performance by establishing substantial
microbial communities.
 To clarify the mechanism of collaboration between MAD
and TAD, further studies of the function of shared micro-
organisms should be conducted using other molecular
301
biology technologies (e.g., metagenomics, microarrays,
single cell genomics).
Acknowledgements
This work was conducted under the framework of Research
and Development Program of the Korea Institute of Energy
Research (KIER) (B4-2474-02) and supported by the Advanced
Biomass R&D Center (ABC) of Korea Grant funded by the
Ministry of Education, Science, and Technology (ABC-
2013059453). The research was partially supported by
BK21 þ program through the National Research Foundation of
Korea funded by the Ministry of Education, Science and
Technology, the Manpower Development Program for Marine
Energy funded by Ministry of Land, Transportation and Mari-
time Affairs (MLTM) of Korean government. This research was
also supported by POSCO and the Korea Institute of Energy
Technology Evaluation and Planning (KETEP) grant funded by
the Korea Government Ministry of Knowledge Economy (No.
2012K130y). This research was a part of the project titled
‘Technology Development of Marine Industrial Biomaterials’,
funded by the Ministry of Oceans and Fisheries, Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2015.01.038.
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