Bioresource Technology 209 (2016) 369379

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Impacts of trace element supplementation on the performance

of anaerobic digestion process: A critical review
Yee Yaw Choong a, Ismail Norli a,, Ahmad Zuhairi Abdullah b, Mohd Firdaus Yhaya c
a

Environmental Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia
School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia
c
Bioresource, Paper and Coatings Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia
b

h i g h l i g h t s

Trace element supplementation improves the anaerobic digestion performance.

Fe, Ni, Co are the most desirable trace elements in the anaerobic digestion system.

Multi-elements supplementation can be more beneficial.

Response of trace element supplementation is uncertain.

Future research should consider crucial aspect of trace element bioavailability.

a r t i c l e

i n f o

Article history:
Received 11 January 2016
Received in revised form 1 March 2016
Accepted 3 March 2016
Available online 10 March 2016
Keywords:
Trace element supplementation
Anaerobic digestion
Metalloenzyme
Methanogenesis
Bioavailability

a b s t r a c t
This paper critically reviews the impacts of supplementing trace elements on the anaerobic digestion performance. The in-depth knowledge of trace elements as micronutrients and metalloenzyme components
justifies trace element supplementation into the anaerobic digestion system. Most of the earlier studies
reported that trace elements addition at (sub)optimum dosages had positive impacts mainly longer term
on digester stability with greater organic matter degradation, low volatile fatty acids (VFA) concentration
and higher biogas production. However, these positive impacts and element requirements are not fully
understood, they are explained on a case to case basis because of the great variance of the anaerobic
digestion operation. Iron (Fe), nickel (Ni) and cobalt (Co) are the most studied and desirable elements.
The right combination of multi-elements supplementation can have greater positive impact. This measure is highly recommended, especially for the mono-digestion of micronutrient-deficient substrates.
The future research should consider the aspect of trace element bioavailability.
2016 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Roles of trace elements in anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Trace element composition of methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Metalloenzymes in methanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Trace elements in hydrolysis, acidogenesis, and acetogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Impacts of single trace element supplementation on anaerobic digestion performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Iron (Fe) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Nickel (Ni) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Cobalt (Co) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Influential factor that causes the impacts variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Impacts of multiple trace elements supplementation on anaerobic digestion performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Crucial consideration: Trace element bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +60 4 6532824; fax: +60 4 6573678.

E-mail address: norlii@usm.my (I. Norli).
http://dx.doi.org/10.1016/j.biortech.2016.03.028
0960-8524/ 2016 Elsevier Ltd. All rights reserved.

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Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

5.1.
Methodology to determine trace element bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Trace elements bioavailability of anaerobic digestion feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Chelating agents to increase trace element bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Research outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

2. Roles of trace elements in anaerobic digestion

Anaerobic digestion has been one of the most yearning and

promising biological technologies for treating a wide variety of
organic wastes with high treatment efficiency. This is because it
enables by-production of biogas as a potential renewable energy
source while managing the waste sustainably at the same time.
These advantages not only have greatly encouraged the practice
of anaerobic digestion worldwide, but also attracted the attention
of researchers to further improve the digestion efficiency with less
problems encountered during the process. This is because anaerobic digestion is a sensitive multi-stages process (hydrolysis, acidogenesis, acetogenesis and methanogenesis) which depends on
susceptible microorganisms to carry out the digestion job (De
Vrieze et al., 2012). One of the influential factors is the presence
of trace elements in the digestion system which mainly function
as the micronutrients. The trace elements must be adequate for
supporting the metabolism of microorganisms in order to maintain
the effective digestion process. Otherwise, the performance of
anaerobic digestion will significantly deteriorate (Osuna et al.,
2003a).
Besides basic macronutrients such as carbon (C), nitrogen (N),
phosphorus (P) and sulphur (S), the anaerobes also require the
growing factor of trace elements at relatively lower concentration.
This aspect has been highlighted by researchers and many studies
have been carried out to demonstrate the importance of trace elements in anaerobic digestion. For example, the strategy of supplementing iron (Fe), nickel (Ni), cobalt (Co), molybdenum (Mo) etc. in
the digestion system had successfully manifested a promotion in
the anaerobic digestion performance with different substrates
including maize silage (Pobeheim et al., 2010), wheat stillage
(Schmidt et al., 2014), food waste (Wei et al., 2014), and so on. In
addition, Fermoso et al. (2009) indicated the fundamental role of
trace elements by summarizing the knowledge of interactions
between trace elements and microbe cells. Due to the natural characteristics of low trace element content, special attention had been
paid by Demirel and Scherer (2011) on the solid organic substrates
such as energy crops, crop residues, and organic fraction of municipal solid waste (OFMSW) by reviewing trace element requirements in their anaerobic digester.
The interest on anaerobic digestion for biogas production is
continuously growing nowadays. Therefore, more substantial
and relevant knowledge should be well documented for future
references. However, despite the vital aspect of trace elements
in the anaerobic digestion system that have been well established, the detail information on their roles and impacts are still
scattered in the literature. Hence, this review paper will firstly
introduce the roles of trace elements in anaerobic digestion, especially from the aspect of methanogenesis for the fundamental
understandings and justifications. Furthermore, this work aims
at compiling the information on impacts of supplementing trace
elements on the performance of anaerobic digestion process
based on the researches which had been done previously and
discuss them critically.

2.1. Trace element composition of methanogens

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Methanogenesis is the final and most critical stage of anaerobic

digestion process ultimately produces biogas, and it is completed
by the strict group of anaerobes called methanogens under the
Archaea domain. Besides of the major elements i.e. C, H, N, P and
S, microbes also highly depend on the content of trace elements
to survive and carry out various cell metabolisms. Therefore, it is
important to have an anaerobic digestion system with sufficient
level of trace elements to ensure the optimal and stable performance. For discovering the required trace elements by methanogens, Scherer et al. (1983) had analyzed the elements
composition of different species and strains of methanogens, and
part of the results are shown in Table 1. Overall, the content of
these elements in methanogens can be summarized in the order
of Fe >> Zn > Ni > Cu  Co  Mo > Mn. This outcome clearly indicates that trace elements indeed play essential roles as the methanogens building elements.
Results in Table 1 are also consistent with earlier findings
reported by Schnheit et al. (1979) who found out that Methanobacterium thermoautotrophicum grew dependently on trace elements
of Fe > Ni > Co = Mo. For further justification, there is another study
that pointed out that trace elements content lower than 30, 4.8,
1.32, 1.13, 0.12 g L1, for Co, Ni, Fe, Zn, Cu respectively started to
limit the growth of methanogen culture in term of cell density
(Zhang et al., 2003). Besides the role as a growing factor, Fe was discovered to be also important in stimulating the formation of cytochromes and ferroxins (Fd) which are vital for cell energy
metabolism. Thus, increasing Fe has been found to enhance the rate
of methane formation by Methanosarcina barkeri with methanol as
the substrate (Lin et al., 1990). Obviously, methanogenesis as well
as the anaerobic digestion process can only be carried out
Table 1
Trace element composition of methanogens. Modified from Scherer et al. (1983).
Methanogen species

Methanobacterium
bryantii
Methanothermobacter
marburgensis
Methanobrevibacter
arboriphilicus
Methanobrevibacter
smithii
Methanosarcina mazei
Methanosarcina
vacuolata
Methanosarcina
thermophila
Methanosarcina barkeri
Methanosarcina barkeri
Methanosarcina barkeri

DSMNo

Content (ppm)
Fe

Ni

Co

Mo

Zn

Mn

Cu

862

1350

150

20

25

250

160

2133

1500

110

10

25

50

<5

<10

744

1300

65

15

40

440

20

861

1100

95

90

25

630

25

2053
1232

720
1600

95
70

35
30

<10
30

170
260

5
15

10
40

1825

1600

150

50

<10

230

<10

800
1538
804

1100
1200
2150

65
60
135

30
25
60

25
45
60

300
370
130

10
10
5

10
35
10

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Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

Table 2
Trace element stimulatory concentration in the batch culture of methanogens. Modified from Takashima et al. (1990).
Trace element

Substrate

Species

Stimulatory concentration (mg L1)

References

Fe
Ni

H2/CO2
Methanol
H2/CO2
H2/CO2
H2/CO2
Formate
Methanol
H2/CO2
H2/CO2
Formate
H2/CO2
Methanol

Methanobacterium thermoautotrophicum
Methanosarcina barkeri
Methanobacterium smithii
Methanobacterium bryantii
Methanococcus voltae
Methanococcus vannielii
Methanosarcina barkeri
Methanococcus voltae
Methanococcus maripaludis
Methanococcus vannielii
Methanobacterium thermoautotrophicum
Methanosarcina barkeri

1.8
0.0059
0.059
0.295
0.12
0.079
0.079
0.79
0.79
18.3
>0.00096a
0.048a

Taylor and Pirt (1977)

Scherer and Sahm (1981)
Diekert et al. (1981)
Jarrell et al. (1982)
Whitman et al. (1982)
Jones and Stadtman (1977)
Scherer and Sahm (1981)
Whitman et al. (1982)
Jones et al. (1983)
Jones and Stadtman (1977)
Schnheit et al. (1979)
Scherer and Sahm (1981)

Co
Se

W
Mo
a

Claimed as optimum concentration (mg L1).

successfully by having obligate concentrations of trace elements at

least more than the stimulatory levels (Takashima et al., 1990).
Cultivation of pure methanogens in the known media had been
commonly applied to determine the stimulatory concentration of
various micronutrients (Table 2). On the contrary, the distinct
requirement of trace elements of the mixed methanogens culture
are not well understood. Compared to cultivate mixed methanogens in a defined medium, the actual wastewater (sewage, industrial, and agricultural) is rather applied to study the role of trace
elements in the anaerobic digestion system. However, the aspect
of microbial communities is relatively unfocused due to time consuming, but more researches are looking into the performance of
anaerobic digestion. This aspect will be critically discussed in Sections 3 and 4.
2.2. Metalloenzymes in methanogenesis
There are three main biochemical pathways involved in
methanogenesis which are aceticlastic, CO2/H2, and methylotrophic pathways. Aceticlastic pathway which uses acetate as
substrate accounts the major contribution of methane formation
(70%) in an anaerobic digestion process, while the remaining 30%
by the CO2/H2 pathway (Mara et al., 2003). The minor methylotrophic pathway will not be discussed in detail in this part as
its working mechanism is an inverse of CO2/H2 pathways. The
working mechanism of aceticlastic and CO2/H2 pathways involved
various metalloenzymes and the trace element requirements of
these enzymes will be different depending on the undergoing
pathway (Glass and Orphan, 2012).
Aceticlastic and CO2/H2 pathways will initially undergo
different reactions before forming either methyl-tetrahydrosarcinapterin (CH3-H4SPT) and methyl-tetrahydromethanopterin
(CH3-H4MPT), respectively. These reactions are participated by different types of enzyme, especially the metalloenzymes. In short,
the trace elements in the metalloenzyme are functioning to form
the active site, cofactor and the structure itself. For instance, the
first metalloenzyme required in the aceticlastic pathway is carbon

monoxide dehydrogenase/acetyl-coenzyme A synthase (Cdh) complex enzyme, which the structure of its subunit CdhA was found to
contain the elements Fe and Ni (Harrop and Mascharak, 2005). On
the other hand, formylmethabofuran dehydrogenase contains two
isoenzymes which need at least one binding element of Mo or
tungsten (W) to initiate the biochemical reaction of CO2/H2 pathway (Hochheimer et al., 1998). Last but not least, the enzyme CH3H4M(S)PT-coenzyme M methyltransferase (Mtr) contains cofactor
5-hydroxybenzimidazolylcobamide (factor III, with element Co)
and the crystal structure of methyl coenzyme M reductase (Mcr)
which has been discovered with two nickel-containing cofactors
F430 as the active sites (Ferry, 2010).
Based on the pathways, the general trace element requirement
trend for metalloenzymes in methanogenesis can be simplified as
Fe >> Ni > Co > Zn = Mo/W (Glass and Orphan, 2012). This order is
predicted without taking consideration of the substrate medium
which will shift the enzyme ratios, and hence alter the respective
trace elements requirement. Therefore, although the fundamental
understanding of methanogenic pathways has been well understood and established, further research is necessary to assess the
possible mechanism and pathway regulatory factors.
2.3. Trace elements in hydrolysis, acidogenesis, and acetogenesis
Acetate is the key intermediate substrate of aceticlastic pathway which has been discussed in the previous section. Acetogenesis, which converts the carbon compounds such as CO2, CO,
methanol and formate to acetate, involves some metalloenzymes
in the reactions. Typically, the formate dehydrogenase (Fdh) and
carbon monoxide dehydrogenase (CODH) are identified by
enzyme-isolating the acetogen Clostridium thermoaceticum
(Ljungdahl, 1986). Table 3 summarized the trace elements in the
metalloenzymes for both acetogenesis and methanogenesis.
Unlike acetogenesis and methonogenesis, the role of trace elements in the stages of hydrolysis and acidogenesis in the anaerobic
digestion process is relatively lacking of research study. Nevertheless, Kim et al. (2003) reported the micronutrients (Fe, Co, and Ni)

Table 3
Trace element in the metalloenzymes.
Enzyme/Cofactor

Process

Trace element(s)

Reaction

References

Formate dehydrogenase
Carbon monoxide dehydrogenase
Hydrogenase
Carbonic anhydrase
Formylmethanofuran dehydrogenase
Methyltransferase/factor III
Methyl-CoM reductase/F430

Acetogenesis
Acetogenesis
Acetogenesis & methanogenesis
Methanogenesis
Methanogenesis
Methanogenesis
Methanogenesis

Fe, Se, W
Fe, Ni, Zn
Fe and/or Ni
Zn
Fe, Mo/W
Co
Ni

HCOOH M CO2 + 2e + 2H+

CO + H2O M CO2 + 2e + 2H+
H2 M 2e + 2H+
+
CO2 + H2O M HCO
3 +H
CO2 + MF ? CHO-MF + H2O
MeOH + CoM ? CH3-CoM + H2O
CH3-CoM + [H] ? CH4 + CoM

Ljungdahl (1986)
Ljungdahl (1986)
Shima et al. (2002)
Glass and Orphan (2012)
Hochheimer et al. (1998)
Ferry (2010)
Ferry (2010)

Me: methyl group.

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Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

were found critical to optimize the process of hydrolysis and acidogenesis as the trace elements supplementation had increased the
COD solubilization and organic acids production. Likewise, Yu
et al. (2015b) highlighted the supplementation of Fe (III) had accelerated the hydrolysis and the acidification process of a thermophilic
anaerobic digestion system. Logically, this can be explained that the
hydraulic and acidogenic bacteria have benefited from trace elements as the growing factor. For the better understanding of the
other roles of trace elements playing in hydrolysis and acidogenesis, it is highly urged for the future research works.
3. Impacts of single trace element supplementation on
anaerobic digestion performance
3.1. Iron (Fe)
Among various of trace elements, Fe is the most commonly
studied element for the supplementation impact on the anaerobic
digestion due to its high requirement based on the fundamental
knowledge. First and foremost, Park and Novak (2013) demonstrated the direct addition of Fe (III) at 1.25% (by weight) into a
sewage sludge digestion system to remove the odour-causing
byproducts. They observed that the problematic and corrosive
hydrogen sulphide (H2S) which had been reduced by more than
65%. H2S generation is reduced due to the FeS precipitation, which
had consumed the metabolite bisulphide (HS) produced by the
sulfate reducing bacteria. One of the reasons which has stunted
the industrial practice of full scale biogas plant is the high capital
cost which delays the turnover period. Hence, the H2S reduction
in generated biogas composition can lower the processing cost
for H2S removal in order to utilize the biogas at the end. Nevertheless, the precipitate FeS was claimed to be controlling the solubility
of the other nutrient metals, such as Ni and Co (Gustavsson et al.,
2013b). Thus the aspect of trace element bioavailability (Section 5)
should be considered when the measure of Fe-dosing is applied.
The leftover intermediates of hydrolysis and acidogenesis reactions such as propionate and butyrate are required to be converted
into acetate before undergoing the methanogenesis. The accumulation of these intermediates will greatly inhibit the overall process
of anaerobic digestion. For this, the method of Fe0 supplementation
had been discovered to be useful in increasing the propionate conversion rate by having more propionate-utilizing and homoacetogenic bacteria (Meng et al., 2013). In fact, the Fe dosing time was
found to be influential on balancing the system of anaerobic digestion, which the 72th-hour after digester start-up was the most
suitable dosing time (Yu et al., 2015b). Yet, this factor has not been
emphasized for the other trace elements.
By considering the additional cost needed in nutrient (Fechemical) addition, the substrate which contains sufficient trace
elements will be favorable to be the alternative supplement. The
Fe-rich activated sludge (1350 mg Fe L1) was claimed to be a suitable co-substrate by De Vrieze et al. (2013) which had significantly
enhanced the methane generation by 326% compared to a
mono-digestion reactor, which was fed with kitchen waste under
mesophilic condition. In addition, the stabilizing effect of Fe-rich
seeding sludge was reported in the same study with relatively
more effective start-up process. Its volatile fatty acid (VFA) concentration also never exceeded 500 mg COD L1, unlike the control
reactor which had failed to perform the digestion at the end.

ing the acetate utilization rate, Ni stimulation has also been found
to have a connection with the predominance of a genus Methanosarcina (Speece et al., 1983). The conclusion of Munk and Lebuhn
(2014) is also in agreement with this statement in which they discovered that the microbial population in a Ni-supplemented
(0.12 mg Ni kg1fresh matter) fermenter with maize feeding had
shifted dominance to Methanosarcina sp. Similar finding has been
reported by Yu et al. (2015a) by using Fe salt instead. This phenomena is obviously beneficial because the community of Methanosarcina sp. is a versatile and it is the key methanogen in the anaerobic
digestion system. It can undergo both methanogenesis pathways
with high tolerance to different stressful living surroundings such
as high level of ammonium (up to 7000 mg L1), and sodium (up to
18,000 mg L1) compared to other species of methanogens (De
Vrieze et al., 2012).
In terms of digestion performance, the presence of Ni can stimulate the degradation rate of acetate (Speece et al., 1983). On the
other hand, Ni alone probably may not significantly improve the
total biogas production, but the supplement of 0.1 mg Ni L1 successfully increased the methane yield by 27% in a maize silagefed batch reactor (Evranos and Demirel, 2015). This achievement
is strongly pursuited for improving the quality of the generated
biogas with a higher calorific value which depends on methane
content. Besides the increased methane generation, the continuous
addition of Ni (0.5 lM) into a methanol-fed upflow anaerobic
sludge bed (UASB) immediately solved the VFA accumulation
problem in the bioreactor and hence, recovered the methanol
degradation up to 99% (Fermoso et al., 2008).
3.3. Cobalt (Co)
Jarvis et al. (1997) reported that Co at a concentration of
0.02 mg L1 was required in order to maintain a stable and efficient
anaerobic digestion performance of grass-clover silage. Apart from
improving the methane production and the acetate degradation
rate (<0.05 g CH3COO L1after Co addition), Co-additive also
allowed a higher reactor working capacity, which was up to
7.0 gVS L1d1 compared to 5.0 gVS L1d1 of the Counsupplemented reactor. These indicated that Co is one of the crucial elements to maintain the stability of the digestion process,
while allowing higher organic loading.
On the other hand, a Co-deprived UASB fed with methanol was
found to have a low methanogenic activity during digestion and
this was probably caused by the shifting pathway of methanogenesis to methylotrophic (Zandvoort et al., 2002). The measure of the
authors to complement the bioreactor with 0.33 lM of Co managed to improve the methanol conversion efficiency of the bioreactor. A relatively higher dosage of 0.84 lM significantly increased
the methanogenic activity of intermediate acetate by maximum
of 75%. Besides stimulating the methanogenesis process, the addition of Co is believed to boost the acetogenesis at the initial stage of
anaerobic digestion (Boonyakitsombut et al., 2002; Florencio et al.,
1993). This is because Co is one of the growing factors of acetogens
(Ljungdahl, 1986). Not forgetting, (sub)optimal dosage is critical to
avoid the accumulation of acetate, which will constrain the overall
anaerobic digestion process (Paulo et al., 2004). From the toxicity
point of view, the concentration of 70 mg L1 free Co and
280 mg L1 of total soluble Co or higher will completely inhibit
the process of methanogenesis (Bhattacharya et al., 1995).
3.4. Influential factor that causes the impacts variation

3.2. Nickel (Ni)

The vital role of Ni in anaerobic digestion has been reported
where Ni is required for the growth of all the methanogens and
the synthesis of cofactor F430 (Diekert et al., 1981). Besides enhanc-

There is a very limited literature material focusing on the

individual impacts raised by a single element except Fe, Ni and
Co. The relevant and representative literature data for single
element supplementation is presented in Table 4. Based on the

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Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

Table 4
Impacts of single trace element supplementation on anaerobic digestion performance.
Trace
element

Substrate

Operation mode
and condition

Additive
concentration

Unit

Fe

Acetate

CSTR
OLR: 0.6 g/L day
Batch, 37 C
CSTR
OLR: 0.6 g/L day
Batch, 37 C
Batch, 37 C
Batch, 37 C

1.0a/
1.060b
100
0.1/
0.201a,b
100
100
0.1

mg L1

5
0.1a/
0.154b
100
100
0.1

mg L1
mg L1

Food wastec
Food wasted
Maize silage

Batch, 37 C
CSTR
OLR: 0.6 g/L day
Batch, 37 C
Batch, 37 C
Batch, 37 C

Food waste
Food wastec
Food wasted
Maize silage

Batch,
Batch,
Batch,
Batch,

37 C
37 C
37 C
37 C

1
6
6
0.05

mg L1
mg kg1TSin
mg kg1TSin
mg L1

Food
Food
Food
Food
Food

Batch,
Batch,
Batch,
Batch,
Batch,

37 C
37 C
37 C
37 C
37 C

5
10
10
10
10

mg L1
mg kg1TSin
mg kg1TSin
mg kg1TSin
mg kg1TSin

Ni

Food waste
Acetate
Food wastec
Food wasted
Maize silage

Co

Mo

Se
W

Food waste
Acetate

waste
wastec
wasted
wastec
wasted

1

mg L
mg L1
mg kg1TSin
mg kg1TSin
mg L1

mg kg1TSin
mg kg1TSin
mg L1

Parameter

Impact (%)

References

AUR
VSS
Methane yield
AUR
VSS
SMP
SMP
TS removal
VS removal
Methane yield
AUR
VSS
SMP
SMP
TS removal
VS removal
Methane yield
SMP
SMP
TS removal
VS removal
Methane yield
SMP
SMP
SMP
SMP

+3822
+1390
+11.3
+2011
+352
+15.4
6.22
+0.2
+0.5
+14.0
+462
+258
+11.2
7.14
No impact
+0.7
+12.4
+42.9
0.230
0.4
3.6
+11.6
+27.2
9.45
+10.7
20.3

Takashima and Speece (1989)

Zhang et al. (2015b)
Takashima and Speece (1989)
Facchin et al. (2013)
Facchin et al. (2013)
Evranos and Demirel (2015)
Zhang et al. (2015b)
Takashima and Speece (1989)
Facchin et al. (2013)
Facchin et al. (2013)
Evranos and Demirel (2015)
Zhang et al. (2015b)
Facchin et al. (2013)
Facchin et al. (2013)
Evranos and Demirel (2015)
Zhang et al. (2015b)
Facchin et al. (2013)
Facchin et al. (2013)
Facchin et al. (2013)
Facchin et al. (2013)

Note: +: higher; : lower by finding comparison of trace element supplemented and unsupplemented/control system.
AUR: acetate utilization rate; TS: total solids; VS: volatile solids; VSS: volatile suspended solids; SMP: specific methane production; CSTR: continuous stirred tank reactor;
OLR: organic loading rate.
a
Minimum requirement to support capacity 30 g L1d1 AUR at 5-day solid/hydraulic retention time.
b
Corrected concentration by taking account of contamination.
c
Inoculated with low concentration of background trace element (mg kg1TS): Ni: 24.2; Co: 2.9; Mo: 4.0; Se: <1.0; W: 2.7.
d
Inoculated with high concentration of background trace element (mg kg1TS): Ni: 47.0; Co: 7.4; Mo: 15.9; Se: 9.1; W: 5.2.

table, all the supplemented trace elements except Mo, have positive impacts on the acetate degradation rate, methane generation
and solid destruction. In a study by Evranos and Demirel (2015),
Mo addition had been demonstrated to have a negative impact
on solid removal, but a higher methane production was observed
at the end of digestion. Obviously, there is a great variation of
impact levels caused by different single trace elements on the
anaerobic digestion performance, and this situation can be
explained by some reasons and factors.
First and foremost, the type of digestion substrates. The simpler
substrate, i.e. acetate in the study of Takashima and Speece (1989)
will have a greater response on the trace element supplementation
compared to the substrate which has a complex matrix (maize
silage; Evranos and Demirel, 2015). Despite the studied parameters, additive concentration and operation mode were different,
the obvious significant differences of the impact levels are sufficient to support this statement.
The factor of trace element background level clearly influences
the impact of supplementation. This can be observed from the findings of Facchin et al. (2013), where the high-trace-element-level
inoculated food waste had a negative impact on the methane production and vice versa (Table 4). Therefore, it is critical to identify
and study the trace elements background level of substrates before
supplementing the trace element to avoid overdosing and deterioration of digestion performance (Plugge et al., 2009). For this, the
regional estimation of the element concentration of substrates will
be desirable, yet this would be infeasible. This is because there was
a high regional variation without a traceable trend in the level of
certain micronutrients by analyzing 1500 samples from different
digesters (Lindorfer et al., 2012).
Moreover, the impact of trace element is believed to be affected
by the operating temperature, typically mesophilic and

thermophilic. According to Takashima et al. (2011), 2.27.8 times

of Fe, Ni, Co, and Zn are needed by a thermophilic system than
the mesophilic for glucose fermentation by having the similar
chemical oxygen demand (COD) input concentration. This is probably due to the general low operational stability of a thermophilic
system which would give a different response, although receiving
the same amount of trace elements. However, the high degree of
substrate destruction of thermophilic system makes the measure
of trace element supplementation worthy to be further studied.
All in all, the supplementation of single trace element of most
trace elements (Fe, Ni, Co, Mo, Se, and W) can effectively improve
the anaerobic digestion performance by stages. Firstly, they support the growth of acetogens (Ljungdahl, 1986), methanogens
(Scherer et al., 1983) with sufficient of metalloenzymes. Then, they
can increase the substrates degradation efficiency, pollutants
(COD, solids) removal and finally improve the biogas/methane production. However, these impacts are not certain, but strongly
affected by many factors such as operational mode and condition,
feed-in substrate, reactor configuration, total background element
concentration, and so on. Due to the complex process of anaerobic
digestion, the regular and certain impact of every single element
cannot be interpreted but depends on a case basis. Anyhow, the
supplementation of necessary trace element with (sub)optimal
dosage will definitely be useful in enhancing the anaerobic digestion performance due to their essential roles and functions.

4. Impacts of multiple trace elements supplementation on

anaerobic digestion performance
By comparing the impact levels of single trace element addition,
the synergism of multiple trace elements supplementation can be

374

Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

observed with a better performance of anaerobic digestion

(Facchin et al., 2013; Zhang et al., 2015b). Besides that, the collaborative supplementation might be needed, otherwise the digestion
performance could be affected negatively. This situation has been
reported by Moestedt et al. (2016) in which Co addition alone
caused a higher acetogenesis rate than methanogenesis. Eventually, the add-on Ni stabilized the system by having the immediate
responses on positive biogas production and VFA elimination (7.0
0 mg L1) within five days. Thus, the correct combination of trace
elements supplementation can be more beneficial. For this, the
interactive effects between/among the elements are crucial to be
figured out which is one of the research gaps to be further
addressed.
As discussed in the previous section, the effects of trace element
supplementation are interrelated and occurred in stages simultaneously. For the foundation, the microbial communities must be
beneficial and consequently improve the anaerobic digestion performance. For example, the addition of Ni/Mo/B and Se/W trace
element solutions could positively influence the archeal population and Methanosarcina sp. respectively and at last, the methane
production efficiency was improved by 715% (Feng et al., 2010).
Moreover, the additive of trace elements resulted in higher biomass content in the digester based on the study by Zhang et al.
(2015a) by applying Fe/Ni/Co mixture. Similarly, Speece et al.
(1983) also suggested that Ni, Fe and/or Co should be supplemented into the digester for achieving high volatile suspended
solids (VSS) contents.
The balanced system between the processes of hydrolysis, acidogenesis, acetogenesis, and methanogenesis are so crucial in
order to have a successful anaerobic digestion system. Especially,
the VFA content should be degraded at the higher rate, or at least
at the rate which can obtain the optimum pH level (6.57.5) for
maintaining the ideal living surroundings of microbes and stability
of the digester. For this, trace elements supplementation was found
to be effective in enhancing the VFA elimination and hence control
the level of it (Climenhaga and Banks, 2008; Ortner et al., 2014a;
Wall et al., 2014; Wei et al., 2014). These situations are usually followed by a greater effectiveness of organic matter digestion. For
instance, the codigestion system of food waste and swine wastewater had a negligible VFA level, low soluble chemical oxygen
demand (SCOD), and higher solid destruction after the addition
of Fe/Ni/Mo/Co (Zhang et al., 2011). Apart from that, a higher
feed-in capacity of digester can be achieved by having an undercontrolled VFA concentration without affecting the digestion performance significantly (Zhang et al., 2015a).
Furthermore, Yirong et al. (2014) reported the cocktail of trace
elements assisted start-up of a thermophilic digester by decreasing
the initial VFA level and also preventing the VFA accumulation during the digestion process. Besides the starting-up, the failed and
unfed digesters were successfully restarted operation with the
addition of trace elements (Bayr et al., 2012; Wei et al., 2014).
On the other hand, the Fe/Ni-deficient reactor, which was wheat
stillage fed had shown a rapid accumulation of VFA content
(Schmidt et al., 2014).
Next, the conversion rate of the immediate substrates such as
acetate, propionate and methanol was found to be stimulated by
supplementing trace elements mixture (Osuna et al., 2003b). As a
result, the improvement of the biogas production in terms of volumetric and/or methane content can be observed (Banks et al.,
2012; Nordell et al., 2016; Zhang et al., 2015b). Table 5 compiles
the findings of the previous works and clearly indicates the
impacts of adding the multiple trace elements on anaerobic digestion process.
In most cases, the strategy of adding trace elements into the
anaerobic digestion system is beneficial. However, these responses
are impossible to be certain, but varied and dependent on many

factors such as the reactor configuration (Patidar and Tare, 2004).

This is due to the complex mechanism of anaerobic digestion and
wide-range variation of operational condition with different types
of substrate. But, this strategy is highly recommended for the
digestion substrates which originally have low trace element contents such as food wastes (Banks et al., 2012; Wei et al., 2014;
Zhang and Jahng, 2012; Zhang et al., 2015a) and stillage
(Gustavsson et al., 2013a) to achieve higher long term process stability of more than 1 year, with a precondition of sustainable
organic loading.
5. Crucial consideration: Trace element bioavailability
The trace elements characterization is the first screening procedure to analyze the nutrients fulfilment in the digester. For this,
most of the studies had characterized the substrates in term of
total metal concentration (Evranos and Demirel, 2015; Qiang
et al., 2013; Zhang and Jahng, 2012). However, the total element
contents can no longer be representative as the bioavailable fraction which can be utilized along the anaerobic digestion process.
This is because there are complex interactions between the liquid
phase and solid phase in the anaerobic digester (Zandvoort et al.,
2006). Especially, the metal-biomass interaction, which involves
the reactions of precipitation, co-precipitation, and adsorption
(Gustavsson et al., 2013a). These reactions can be triggered by
the occurrence of sulphide chemistry, which had been claimed as
the main regulator of the trace elements bioavailability in the
anaerobic digestion system (Fermoso et al., 2009). For instance,
the metal sulphide of FeS is well known to potentially adsorb
and/or co-precipitate Ni and Co, and then forms the complexes of
Fe-Ni/Co-S (Gustavsson et al., 2013b; Shakeri Yekta et al., 2016).
Theoretically, these interactions will reduce the bioavailability
fraction in the form of free metal for the uptake by microbial communities inside the digester.
5.1. Methodology to determine trace element bioavailability
Sequential extraction is the most common applied methodology
to determine the metal bioavailability. The basic idea of this
method is to subsequently extract the metals by fractions using
the correspond extracting reagents, which have the rising metal
leaching power. In general, the stronger the metal leaching power
of the applied extractant, the lower the bioavailability of the
extracted metal. Therefore, the method of sequential extraction is
highly depending on the extracting conditions, such as the extraction time, reagents pH and concentration.
Besides of time consuming, sequential extraction has the irregular and divergent issues. These problems have greatly discouraged the aspect of trace elements bioavailability to be taken
consideration in the anaerobic digestion studies. Actually, there
are many existing sequential extraction schemes to extract different numbers and types of metal fraction by using various of extractants. Filgueiras et al. (2002). had comprehensively reviewed the
sequential extraction schemes which had been applied for metal
partitioning in environmental studies (soil, dust, sediment, and
so on). While, for the anaerobic digestion study, the Tessier scheme
(Tessier et al., 1979) and its modifications had been commonly
applied (Thanh et al., 2016).
For example, Ortner et al. (2014b) had modified the Tessier
scheme to identify the bioavailability of trace elements in the
anaerobic digestion system, which skipped the step of sample drying before partitioning the metal fractions of soluble, exchangeable, carbonate, organic/sulphide, and residual. By applying this
modified method, Ortner et al. (2015) concluded that the concentration of bioavailable (soluble and exchangeable) fractions

Table 5
Impacts of multiple trace elements supplementation on anaerobic digestion process.
Substrate

Operation
mode and
temperature

Operation
condition

Trace element
and added
concentration

Unit

Findings

References

Food waste

Semicontinuous,
300 mL digester,
37 C

OLR:
2 gVS L1 d1
HRT: 30 days

Fe: 100
Co: 1
Ni, Mo: 5

mg L1

Methane yield +18.7%

Methane content +3.7%
VS removal +6.5%

Zhang
et al.
(2015b)

OLR:
4 gVS L1 d1
HRT: 20 days

Methane yield +24.5%

Methane content +4.5%
VS removal +8.0%
&

OLR:
6 gVS L1 d1
HRT: 15 days

&

Unsupplemented digester failed and stopped operating.

Supplemented digester performance slightly deteriorated:
Methane yield by 3.95%
Methane content by 1.2%
VS removal by 3.2%

Continuous,
12 L CSTR,
55 C

OLR:
6.8 kgCOD m3d1

Fe: 10
Co, Ni: 1

mg L1

Biogas production +12.7%

Methane content 1.1%
Total solids removal + 3 %
Volatile solids removal +6%
Total COD removal +15%
Carbohydrate removal +11%

Qiang et al.
(2013)

Food waste

Semicontinuous,
4.5 L digester,
37 C

OLR:
1.0 to
3.0 gVSL1d1

Fe: 5
Co, Ni: 1

mg L1

SMP +5.1%

Zhang
et al.
(2015a)

&

OLR:
4.0 gVS L1 d1

&

N-rich slaughterhouse waste (TKN:

11 g kg1)

Continuous,
7 L CSTR,
37 C

OLR:
5.5 gVS L1 d1

Fe: 5
Co, Ni: 1
Se: 0.2

OLR:
2.5 kgVS m3d1

Trace element
solution:
Ni, Co, Cu, Mn, Zn,
Mo, Se, B

n.a.

Manure and industrial waste

8 L CSTR,
39 C

OLR:
3.0 kgVS m3d1
OLR:
3.3 kgVS m3d1

&

Additional of element Se recovered the digestion system by reducing VFA: 9000 to

1200 mg L1 (by 86.7%)

&

Total VFA reduced from 10,000 to <700 mg L1 (by 93%)

SMY increased

&

Trace element
solution:
Fe, Co, Ni, Se, W

n.a.

Ortner
et al.
(2014a)

&

SMY reduced from 340 to 170 Nm3 tCOD1 (by 50%) due to the ammonia inhibition.
Total VFA <3500 mg L1

&

VFA reduced from 1.7 to 0.4 g L1 (by 76.5%)

&

Biogas production rate +24%

Biogas/methane yield +10%

&

OLR:
1.8 kgVS m3d1
NH3-N:
8.5 g kg1

Sharp drop of methane yield in unsupplemented digester

SCOD: 39,000 mg L1
VSS: 1.653 g L1
No significant deterioration of SMP and VMPR in supplemented digester
SCOD:<6500 mg L1
VSS: 2.073 g L1

&

Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

High solid food waste (TS: 14.3%)

Nordell
et al.
(2016)

(continued on next page)

375

376

Table 5 (continued)
Substrate

Operation
mode and
temperature

Operation
condition

Trace element
and added
concentration

Unit

Grass silage

Semicontinuous,
4 L CSTR,
37 C

OLR:
4.0 kgVS m3d1
HRT:
19 days

Fe: 74.4
Co: 0.13
Ni: 2.48

mg L1

Semicontinuous,
4.5 L digester,
37 C

Stop feeding

Trace element
solution:
Fe, Ni, Co

n.a.

Food waste

Findings

Wall et al.
(2014)

&

SMY increased from 360 to 404 LCH4kgVS1 (by 12%)

Accumulation of propionic acid was eliminated
Acetic acid concentration declined to low values

&

Total VFA reduced from 30,470 to 2260 mg L1 (by 92.6%) within 2 weeks

Wei et al.
(2014)

&

High and stable SMP and VMPR

Stop feeding

Trace element
solution:
Co, Ni, Se, W

&

Accumulation of total VFA is much more less than (91.3%) the control reactor, which
was solely supplied with Fe

&

Higher methane yield (7.46%) compared to the control reactor

&

VFA accumulated from <500 to about 4000 mg L1

mg L1

&

Temporal drastic reduction of VFA

1

&

VFA reduced from 10,000 to <500 mg L1

&

VFA accumulated once the OLR increased at the 96th day, yet the concentration of VFA
had decreased afterwards, which was at 148th day by 25%
Methane yield slightly improved from 439 to 446 mL CH4 gVS1
added (by 1.59 %)

n.a.

Restarted with
OLR:
1.5 kgVS m3d1
Food waste with high ammonia
concentration (56.1 gNH3H L1)

Semicontinuous,
4 L digester,
36 C

OLR:
3 to 4 gVS L1 d1

Multi-element
supplementation
stopped
Se: 0.03

Food waste

Semicontinuous,
150 mL digester,
36 C

OLR:
5 gVS L1 d1

Se: 0.2
Co, Ni: 1.0

mg L

OLR:
2.2 to
2.3 gVS L1 d1

Co: 2.0
Mo: 5.0
Ni: 10.0
Fe: 100

mg L1

&

OLR:
3.6 to
4.5 gVS L1 d1

&

The insignificant increases of OLR did not accumulate VFA yet it reduced from 597 to
471 mg L1 (by 21.1 %)

OLR:
6.6 gVS L1 d1

&

Drastic waste loading caused the increasing of VFA (615 mg L1) but it was maintained
at a low level which is below 960 mg L1.
Slight deterioration of methane yield by 8.33%

&

Organic fractions of municipal solid

waste and slaughterhouse waste
(OFMSW + SW)

Semicontinuous,
8.5 L CSTR,
38 C

OLR:
4.0 gVS L1
(3.25 OFMSW
+ 0.75 SW)

Co: 0.5
Fe: 400

mg L1

&

&
&

Co: 0.5
Fe: 400
Ni: 0.5

mg L

1

&
&

Additional of Co surprisingly caused a higher VFA accumulation with 7 g L1, compared to the control reactor, which only supplemented with Fe with VFA concentration 23 g L1
Increasing of VFA followed with a lower rate of biogas production
Lower alkalinity (10.8%) compared to control reactor.

Bayr et al.
(2012)

Banks et al.
(2012)

Zhang and
Jahng,
2012

Moestedt
et al.
(2016)

Administration of Ni showed immediate VFA depletion from 7 to 0 g L1 within 5 days

Combination of Co and Ni is more beneficial than the addition of Ni only with the presence of Fe due to 8% higher of SMP

Note: +: higher; : lower by finding comparison of trace elements supplemented and unsupplemented/control system.
n.a: not applicable; OLR: organic loading rate; HRT: hydraulic retention day; VS: volatile solids; VSS: volatile suspended solids; (S)COD: (soluble) chemical oxygen demand; VFA: volatile fatty acid; SMY: specific methane yield;
SMP: specific methane production; VMPR: volumetric methane production rate; CSTR: continuous stirred tank reactor.

Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

Semicontinuous,
10 L CSTR,
35 C

&
&

Restarted with
OLR:
4.0 gVS L1 d1
Swine slaughterhouse waste

References

Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

directly impacted the anaerobic digestion performance. Hence, the

aspect of trace element bioavailability should be taken account in
the trace elements characterization. Yet, most of the reported concentration of trace element(s) additive did not include this aspect.
Undeniably, these findings remained reliable. However, a discrete
range of optimum trace element additive concentration could not
be obtained from these results when different digestion conditions
and substrates were applied.
5.2. Trace elements bioavailability of anaerobic digestion feedstock
Ortner et al. (2015) had partitioned the trace elements of the
slaughterhouse waste for the characterization purpose before
undergoing the anaerobic digestion. The results indicated the trace
element Fe, Zn, Ni, and Co had a predominant residual fraction
(67%, 45%, 53%, and 43% respectively) which is theoretically
unavailable for the microbial uptake. While, Mo was found to be
mostly (79%) bonded in the form of organic/sulphide. By the trace
elements (Ni, Co, Mo, and Zn) amendment, their absolute amount
of the soluble and exchangeable fractions had significantly
increased. Surprisingly, this trend was not observed for their residual factions, but remained relatively constant over the time. Ultimately, the strategy of trace elements supplementation had
improved the overall performance of the anaerobic digestion.
Instead of dosing the micronutrients into the digester, the existing trace elements content in the feedstocks should be well utilized. For example, the strategy of co-digesting the substrate,
which is originally rich with the trace elements (i.e. sewage sludge,
and livestock manure) can be employed. Zhang et al. (2011) had
reported the trace elements in the co-substrate (swine wastewater) had enhanced the anaerobic digestion of food waste, with a
higher methane yield and without VFA accumulation. However,
these improvements by co-digestion have not been explained in
term of trace element bioavailability, but generally focused on
the reasons of C and N balancing, and toxicants dilution.
5.3. Chelating agents to increase trace element bioavailability
The rationale of the relationship of trace element bioavailability
and the digestion performance justifies further research works to
increase the elements bioavailability by applying the chelating
agents. Examples of these agents are nitrilotriacetic acid (NTA)
(Hu et al., 2008), ethylenediaminetetraacetic acid (EDTA)
(Vintiloiu et al., 2013), and ethylenediamine-N,N0 -disuccinic acid
([S,S]-EDDS) (Zhang et al., 2015b). There were positive outcomes
from these studies which had improved the methane production
due to the improvement in the element bioavailability. However,
Aquino and Stuckey (2007) reported that the presence of EDTA
reduced the methane production rate.
Nevertheless, Zhang et al. (2015b) had clearly stated the environmental and human health risk of EDTA and NTA application,
respectively. In details, the non-biodegradable EDTA may intensify
the water pollution due to the continued release of heavy metal
organic species. While, NTA was criticized because of its carcinogenicity. Hence, the serious secondary problems will be induced,
when the anaerobically digested wastes which had been supplemented with these synthetic chelators are disposed. Consequently,
[S,S]-EDDS was introduced by Zhang et al. (2015b) as a green synthetic chelator. However, this must be further assessed comprehensively about its risk of application to human and the
environment.
Besides of the synthetic chelating agents, Thanh et al. (2016)
had also reviewed the microbes-releasing chelating agents in the
anaerobic digestion system such as soluble microbial products
(SMPs) and extracellular polymeric substances (EPS). These organic
compounds will naturally increase the trace elements bioavailabil-

377

ity through metal binding and complexation. Yet, it is more proper

and acceptable to enhance the microbial activities instead of supplementing SMPs and EPS into the digester. In order to make sure
the effectiveness and cost feasibility of the chelating agent for
improving the performance of anaerobic digestion, more comprehensive studies are needed.

6. Research outlook
The global effort to strengthen the energy security requires the
support of the relevant scientific researches. Same goes to the
anaerobic digestion, in order to further mature this promising
renewable energy production technology. In this paper, the aspect
of trace elements is discussed and regarded. The role of trace elements in anaerobic digestion had been well understood for the
stages of acetogenesis and methanogenesis, but not for the hydrolysis and acidogenesis process. For fundamental understanding, the
possible regulatory factors that would alter the trace elements
requirement are urged to be investigated. Not forgetting, the interactive relationships between/among the trace elements within the
anaerobic digestion system are required to be evaluated as well.
There is a great challenge to determine a discrete range of trace
elements requirement for the anaerobic digestion process which
involved the complex substrates. One of the reasons is the great
divergence of the background concentration of trace elements content. Therefore, the trace elements bioavailability of the substrates
must be characterized and assessed, especially for the trace elements supplementation study. Only then, the findings such as
the (sub)optimum dosage can be referred and applicable for the
other different substrates. Yet, the anaerobic digestion feedstocks
have not been emphasized for their trace elements bioavailability
compared to the environmental samples for the toxicity
assessment.
There are many researches had been carried out which aimed to
achieve better performance of anaerobic digestion, such as by the
strategies of pretreatment, co-digestion and bioreactor modification. However, these studies had been discussed by focusing on
the well known factors (i.e. biodegradability, C/N ratio, biomass
retention, etc.). Thus, the consideration of trace elements bioavailability in the anaerobic digestion studies is definitely a research
breakthrough. For this, the sequential extraction scheme is crucial
to be uniform and improved. The scheme will be favoured when it
can be easier to partition the representative and relevant metal
fractions with high recovery, but requires low cost and less time.

7. Conclusions
The measure of trace elements supplementation is effective to
improve the performance of anaerobic digestion process by applying necessary (sub)optimum dosage. The positive impacts include
the longer term digester stability with greater organic matter
degradation, low VFA level, and higher biogas production. However, these impacts are uncertain, but can be affected by many factors. Fe, Ni, and Co are the most desirable trace elements. Single
and multiple trace element(s) addition have presented the overlapping positive impacts, but with a greater level by the latter one.
The future researches should be carried out with the consideration
of trace elements bioavailability.
Acknowledgements
The authors gratefully acknowledge Universiti Sains Malaysias
Research University Individual (RUI) Grant (1001/PTEKIND/814239) for the financial support.

378

Y.Y. Choong et al. / Bioresource Technology 209 (2016) 369379

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2016.03.
028.
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