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Stimulation and inhibition of anaerobic digestion by nickel and cobalt: a rapid assessment using the resazurin reduction assay Jian Lin Chen, Terry W.J. Steele, and David C Stuckey Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03522 • Publication Date (Web): 22 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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Environmental Science & Technology

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Manuscript prepared for publication in ES&T

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Stimulation and inhibition of anaerobic digestion by

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nickel and cobalt: a rapid assessment using the resazurin

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reduction assay

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† Jian Lin Chen

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Terry W.J. Steele*,

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David C. Stuckey*,

‡

†, §

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† Nanyang Environment & Water Research Institute, Advanced Environmental Biotechnology

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Centre, Nanyang Technological University, Singapore 637141

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‡ School of Materials Science & Engineering, College of Engineering, Nanyang

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Technological University, Singapore 637141

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§

Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK

KEYWORDS: resazurin; trace metals; stimulation; toxicity; anaerobic digestion

ACS Paragon Plus Environment

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ABSTRACT

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Stimulation of anaerobic digestion by essential trace metals is beneficial from a practical

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point of view to enhance the biodegradability and degradation rate of wastes. Hence, a quick

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method to determine which metal species, and at what concentration, can optimize anaerobic

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digestion is of great interest to both researchers and operators. In this present study, we

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investigated the effect of nickel (II), cobalt (II), and their mixture, on the anaerobic digestion

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of synthetic municipal wastewater. Using a volumetric method, i.e. measuring methane

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production over time, revealed that anaerobic digestion was stimulated by the addition of 5

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mg L-1 nickel (II), and cobalt (II), and their mixture in day(s). However, using a novel

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resazurin reduction assay, and based on its change in rate over time, we evaluated both

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inhibition at 250 mg L-1 nickel (II) and cobalt (II), and also the stimulatory effect of 5 mg L-1

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nickel (II), and cobalt (II), and their mixture, in just 6 hours. By investigating the dynamic

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distribution of these metals in the liquid phase of the anaerobic system and kinetics of

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resazurin reduction by nickel spiked anaerobic sludge, the concentration of nickel (II) on

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anaerobic digestion performance was profiled. Three critical concentrations were determined;

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stimulation starting (around 1 mg L-1), stimulation ending (around 100 mg L-1) and

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stimulation maximizing (around 10 mg L-1). Hence, we propose that the resazurin reduction

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assay is a novel and quick protocol for studying the stimulation of anaerobic bioprocesses by

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bioavailable essential trace metals.


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TOC/Abstract art


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INTRODUCTION

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Heavy metals are common in municipal and industrial effluents, at times causing negative

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effects (toxicity) in biological treatment plants.1,2 However, trace amounts of selected

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metals/elements have been reported to affect the biodegradation and degradation rate of

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wastes beneficially,3,4 while supplementation of small amounts of trace metals has been

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implemented to increase their bioavailability, leading to enhanced biodegradation rates of

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both solid 5 and liquid wastes 6 over time in anaerobic digestion. Some trace metals (such as

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Fe, Co, Ni, Mn) are considered as “essential” elements for microbial growth in anaerobic

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digesters, functioning as protein stabilizers and osmotic balance controllers across various

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microbial membranes, and as catalysts for biochemical reactions, etc.7 A number of studies

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have suggested that the effect of “essential” trace metals on microorganisms can be divided

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into three zones depending on their concentration; increasing stimulation, decreasing

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stimulation, and toxicity.7 However, it was difficult to say exactly what concentration of the

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essential trace metal could cause stimulation or inhibition, and which metals were toxic at

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what concentration, because so many of the results were obtained using different methods,

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and reported in very different ways.8 Hence, the range reported is wide in terms of threshold

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concentrations that result in inhibition or stimulation by the various essential trace metals.7,9

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In addition, the bioavailability of these “essential” trace metals in bioreactors depends on the

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solution chemistry (e.g., pH, anions, cations, presence of complexing agents) which typically

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have a strong influence on interactions between metals in the solid and liquid phases, the

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transport of free metal ions, facilitated diffusion with the assistance of chelating complexes,

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and metal speciation; while proton-mediated transport through the cell membrane are

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assumed to be the main mechanism for metal uptake by microorganisms.7,10 Oleszkiewicz and

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Sharma suggested that even full bioavailability would not necessarily mean that the required

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metals are utilized by the bacterial population7; only when the metal ion enters the cell can it


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really be available for metabolism. However, the large number of chemical (complexation,

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precipitation, adsorption) and biological interactions makes the system complex and difficult

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to understand and control. In practice, it is important to understand metal bioavailability to

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design optimal dosing strategies for anaerobic digesters, and in cases of metal limitations, the

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exact dose and type of essential trace metals is critical to ensure suitable bioavailability,

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leading to optimal bioreactor performance. Therefore, successful stimulation of anaerobic

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digestion by specific essential trace metals, and determining their critical concentrations

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beyond which stimulation becomes inhibition, is important. Given the multivariate

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complexity of these systems, a biological assay which is quick, simple, cheap and accurate

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would enable the field to progress more rapidly.

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Both nickel and cobalt belong to the so-called “essential” trace metals.11 Confirmation of the

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interrelationship between nickel and cobalt is confirmed by the fact that the microbial

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resistance genotypes for both metals are usually present in the same plasmid of many

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microorganisms.12,13 Also, similar biochemical transport mechanisms through microbial

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membranes have been identified for both species.14 Identified as a key component in a

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number of enzymes, nickel participates in important metabolic reactions, such as; hydrogen

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metabolism, methane biogenesis, and acetogenesis.15 Cobalt is another important cofactor in

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Vitamin B12-dependent enzymes,16 and an indispensable component in a number of

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enzymes.17 It has been established that at relatively low concentrations (~10 mg L-1), both

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species can stimulate both aerobic and anaerobic microbial growth, while they have toxic

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effects at higher concentrations (>100 mg L-1).14,18 Therefore, determining these critical

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concentrations of nickel and cobalt for stimulating/inhibiting anaerobic or aerobic

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bioprocesses is critical when optimising these processes. A number of methods have been

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proposed for measuring metal toxicity in activated sludge systems,19 such as the inhibition of;

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enzymatic activity, bacterial respiration and kinetics, and the dynamics of microbial


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community evolution, and for the stimulation of anaerobic digestion by trace metals, such as

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maximum specific methanogenic activity 6 and biochemical methane potential 4. Generally,

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most of these methods are often too slow (days) for the optimal detection of sudden changes

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in anaerobic digestion activity due to metals, and cumbersome in terms of the number of

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variables that can be assessed.19

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Resazurin, which is typically formulated into in-vitro toxicology assay kits (i.e.

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AlamarBlue®), can be used as an oxidation-reduction indicator which results in colorimetric

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changes, and a fluorescent signal in response to intercellular metabolic activity 20 based on the

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reduction of resazurin to resorufin, and finally to dihydroresorufin (Scheme 1). Our previous

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studies on assessing toxicity in anaerobic digestion has demonstrated the sensitivity and

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efficiency of the resazurin reduction assay for detecting organic toxicants such as

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chlorophenols,9 and a model was developed to describe resazurin reduction in anaerobic

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digestion.21 In this present study, we further extend the applicability of this technique by

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investigating the stimulation and inhibition of anaerobic digestion by nickel, cobalt and their

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mixture on anaerobic digestion, and by comparing these results to the methane production test,

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we proposed that the resazurin reduction assay is a novel and more efficient method to study

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the stimulation of anaerobic digestion with bioavailable trace metals.

Scheme 1 105

METHODS

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Chemicals. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide), CoCl2·6H2O,

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NiCl2·6H2O were of high purity and sourced from Sigma-Aldrich, Singapore. Other reagents

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and chemicals used were the highest available purity, and were also obtained from Sigma-

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Aldrich, Singapore.

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Anaerobic Sludge. Anaerobic sludge was obtained from municipal wastewater treatment


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plant digesters and grown in a 1 L laboratory continuously mixed reactor which was operated

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at ~30 °C as the seed for the trace metal tests. The reactor was fed weekly using fill-and-draw

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mode with a synthetic municipal wastewater9,22 (adjusted to pH 7) without trace metals

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consisting of peptone (0.2 g L-1), beef extract (0.14 g L-1), urea (0.01 g L-1), CaCl2·2H2O

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(0.004 g L-1), MgSO4·7H2O (0.002 g L-1), K2HPO4 (0.011 g L-1), NaCl (0.007 g L-1), NaHCO3

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(0.3 g L-1) and acetic acid to give a final COD (chemical oxygen demand) of around 2 g L-1.

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Effect of Spiked Ni2+ and Co2+ on Anaerobic Digestion: Gas Production, Resazurin

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Reduction Assay and Metals Distribution. One hour after feeding the seed reactor, 40 mL

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of fresh anaerobic sludge was taken and mixed with 40 mL of autoclaved synthetic municipal

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wastewater in a 100 mL serum bottle to result in a final VSS of 1.4 g L-1. After being purged

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with N2 for 1 min and sealed with a rubber stopper, a specific amount of CoCl2 or NiCl2

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solution in DI water was spiked into the serum bottles, yielding metal concentrations of 5 and

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250 mg L-1, and mixtures of 2.5 mg L-1 each; a control experiment without metals was also set

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up. Triplicate experiments for each test were carried out in an incubating shaker at 150 rpm

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and 37°C. At 0, 3, 6, 9, 12, 24, 48 and 72 hours the gas volume was measured using calibrated

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wetted glass syringes, and the composition of the gas analyzed using a gas-chromatograph

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(GC- Shimadzu GC 2010) equipped with a TCD. At the same time as for the gas volume

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reading described above, 0.35 mL of mixed anaerobic sludge was taken from the serum

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bottles and added to a 96 well microplate with resazurin (25 mg L-1) under anaerobic

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conditions; the microplate was sealed immediately using sterile film. Fluorescence was read

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once every 1 min for 60 min by an Infinite M200 Pro microplate reader (TECAN, Singapore)

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with TECAN i-control software at λex=530 nm and λem=590 nm, and a gain of 30 according to

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our previous study 9. Meanwhile, at 0, 1, 3, 6, 9, 12 and 24 hours, 5 mL of anaerobic sludge

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was taken and centrifuged at 12000 rpm for 10 min. After filtering through 0.45 µm filters

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and acidifying with HNO3 to 1%, the Ni2+ and Co2+ in the supernatant were measured using


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microwave plasma atomic emission spectrometry (MP-AES; AGILENT model 4200).

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Triplicate serum bottles were also set up with Ni2+ spiked at 0, 0.1, 0.5, 5, 15, 50, 100, 250

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and 1000 mg L-1; after 6 hours 0.35 mL of fresh anaerobic sludge was taken for the resazurin

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(25 mg L-1) reduction assay described above.

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Characterizing Stimulation of Anaerobic Digestion with the Resazurin Reduction Assay.

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Based on a pseudo-first order reaction rate, our previous study developed a model, ሾ‫ܤ‬ሿ =

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ሾ‫ܣ‬ሿ଴

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culture, and mixed sludge under anaerobic conditions,21 where [B] is the resorufin

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concentration linearly proportional to the fluorescent signal, [A]0 is the initial resazurin

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concentration reacting in the cells, k1 and k2 are the reaction rate constants for resazurin to

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resorufin, and resorufin to dihydroresorufin, respectively, and [M] is the total concentration of

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metabolites in the cells to reduce resazurin, which can represent the activity of the

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microorganisms. By setting the 1st derivative of the model equal to zero, the optimum time for

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maximizing produced B (resorufin) is ‫ݐ‬௕௘௦௧ =

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[M]. Therefore, the change in activity of the anaerobes between two sampling points (times a

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and b) can be described by the shift of this optimum time, i.e., ∆tbest = tbest a – tbest b. Hence in

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this present study, the change in activity of microorganisms in anaerobic sludge because of

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spiked trace metals can be used to predict stimulation/inhibition of anaerobic digestion by

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∆tbest.

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Statistical Analysis. The differences in methane production and ∆tbest between the control and

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each metal spiked system at the same sampling time/range were determined using a two-

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sample t-test.

௞భ ௞మ ି௞భ

(݁ ି௞భሾெሿ௧ − ݁ ି௞మ ሾெሿ௧ ), to describe the process of resazurin reduction in both pure

ೖ ୪୬ భ ೖమ

, which is inversely proportional to

ሾெሿ(௞భ ି௞మ )

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RESULTS and DISCUSSION


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Spiking Ni2+ and Co2+ Results in a Significant Stimulation of Methane Production after

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24 Hours. Methane production is a useful indicator when monitoring an anaerobic digester

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suffering from toxicity.23,24 In this study, methane production was also employed as a

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metabolic indicator of trace metals on anaerobic digestion. Fig. 1A shows the volume of

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methane produced every 3 hours in the first 12 hours of the experiments, with and without the

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addition of cobalt, nickel, or their mixture. Comparing methane production to the unspiked

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controls, the inhibition by 250 mg L-1 of spiked Ni2+/Co2+ was obvious; in the first 12 hours

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there was no significant increase in methane production, except in the first 3 hours of the

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experiment with 5 mg L-1 of Ni2+ spiked (Fig. 1A-b), and the 2.5+2.5 mg L-1 Ni2+-Co2+

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mixture (Fig. 1A-c), where there was a significant increase. However, this observation cannot

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prove that the addition of 5 mg L-1 Ni2+, or a mixture of Ni2+ and Co2+, stimulated the

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anaerobic process because methane production in the following 3 sampling times, e.g. 6, 9

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and 12 hours, was not statistically significantly higher than the control. However, when we

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measured the daily methane production rate (Fig. 1B), with the more obvious inhibiting in the

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250 mg L-1 Ni2+ or Co2+ spiked systems, it was found that the volume of methane produced in

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the 5 mg L-1 Ni2+ (Fig. 1B-b), or mixture of Ni2+ and Co2+ (Fig. 1B-c) spiked system was more

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than that in the control from the first day, and for the 5 mg L-1 Co2+ spiked system (Fig. 1B-a),

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that time occurred on the second day of the experiment, meaning that by comparing methane

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production we can tell whether certain trace metal concentrations stimulate anaerobic

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digestion after at least one day.

Figure 1 180

Biogas is the fundamental end-product of anaerobic digestion, and most investigations have

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traditionally focused on biogas measurements to assess metabolic activity and inhibition.

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Although many other methodologies are often used in parallel with gas measurements, e.g.

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the depletion of the concentration of the test substrate, the accumulation of some


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intermediates such as short chain organic acids, the formation of hydrogen,25 biogas

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production is by far the preferred parameter for monitoring the activity of the sludge as a

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whole, particularly when the test material is complex and difficult to measure accurately.

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Biochemical methane potential (BMP) is a measure of the methane that can be produced

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biologically from a given waste, sludge, or substrate, and is used to determine the methane

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generation potential of each waste.23 In contrast, the anaerobic toxicity assay (ATA) was

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developed to predict the likely effect of a potential toxicant on biogas and methane

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production, but does not show microorganism acclimation to the toxicant, or the effect of

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toxicant build-up in the biomass.23 Using these volumetric methods, it takes day(s) to

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determine the inhibitory/stimulatory effect of trace metals on methane production.2,4–6 The

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present study also measured the volume of methane produced, and although the inhibitory

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effects of Co2+ and Ni2+ spiked at 250 mg L-1 could be determined within 6 hours (Fig. 1A-a,

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b), the stimulatory effect of Co

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1

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1B-b, c). In addition, in some cases the outcome of the assay was strongly dependent on the

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rate limiting step, for example, if the test material was particulate, then the hydrolysis step

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was generally rate limiting;26 whereas if it contained a highly proteinaceous fraction, the

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methanogenic step can be slowed down by the accumulation of acids resulting from the

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fermentation, and consequently is likely to be rate limiting.

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Resazurin Reduction Assay Allows Assessment of the Stimulation by Ni2+ and Co2+ in ≤ 6

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hours. With Co2+- The kinetics of resazurin reduction by anaerobic sludge with two Co2+

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spiked levels, i.e. 5 and 250 mg L-1, did not show any significant difference (Fig. 2A)

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according to the estimated tbest of around 147 min for all three systems including the unspiked

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control. In the following sampling time periods, i.e. 3, 6, 9 and 12 hours (Fig. 2B-2E,

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respectively), the spiked 250 mg L-1 Co2+ did not change the kinetics of resazurin reduction

2+

(5 mg L-1), Ni2+ (5 mg L-1) and their mixture (2.5+2.5 mg L-

) could only be determined by methane production after one day (Fig. 1B-a) or two days (Fig.


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significantly, suggesting that 250 mg L-1 Co2+ inhibited the activity of sludge permanently.

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This observation was similar to the results measured by the volumetric method described

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above. However, compared to the control, although the spiked 5 mg L-1 Co2+ slowed down the

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resazurin reduction kinetics, i.e. tbest 3 hours= 75 min (Fig. 2B), tbest 6 hours= 38 min (Fig. 2C),

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tbest 9 hours= 30 min (Fig. 2D) and tbest 12 hours= 30 min (Fig. 2E), with this optimum time

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shifting left, it suggested that the experimental system was recovering from temporary

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inhibition, and this “shift” speed could be an index of the potential bioactivity of anaerobes.

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Fig. 2F shows a summary of the optimum time (tbest) shift every 3 hours, in which it can be

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seen that the ∆ tbest 0-3 hours of the control was greater than that of the 5 mg L-1 Co2+ spiked

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system, while after this time the ∆ tbest of 3-6, 6-9 and 9-12 hours were all significantly greater

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than that of the control, suggesting a higher bioactivity with 5 mg L-1 spiked Co2+. This higher

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potential bioactivity could lead to a faster biogas production rate, which actually has been

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observed empirically by direct methane volume quantitation, as described above. The

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volumetric measurement took 2 days to see the stimulatory effect on biogas production (Fig.

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1B-a), while using the resazurin reduction assay, stimulation by 5 mg L-1 of Co2+ was detected

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within 6 hours.

Figure 2 225

As one of the essential trace metals for microorganisms, cobalt has been reported to show

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different effects on anaerobic processes depending on the type of substrate, and hence

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stimulatory and inhibitory concentrations were found to be between 0.03 and 19 mg L-1, 35

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and 950 mg L-1 (100% inhibition), respectively.5 Cobalt is the central metal ion in corrinoids

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which are present in methylotrophic methanogens and acetogens, e.g., in methanogens;

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corrinoids are involved in methyl transfer from methanol to methyl coenzyme M, the common

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precursor of methane from all substrates.27 In acetogens, corrinoids participate in the

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formation of acetyl coenzyme A, the precursor intermediate of acetate and cell synthesis.28


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However, the content of corrinoids in anaerobic bacteria varies greatly among species and

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substrate utilized, resulting in different stimulatory and inhibitory concentrations for different

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anaerobic processes.29 Hence, rapidly determining the optimal concentration of Co2+ for a

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given anaerobic system would enable engineers to enhance process performance.

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With Ni2+- Permanent inhibition with 250 mg L-1 of spiked Ni2+ was also obtained by

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studying the resazurin reduction kinetics (Fig. 3), and resulted in the same observations as

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with the 250 mg L-1 Ni2+ spiked system measured by methane production (Fig. 1A-b). In

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addition, 5 mg L-1 of spiked Ni2+ slowed down the resazurin reduction kinetics compared to

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the control, i.e. tbest 3 hours= 83 min (Fig. 3B), tbest 6 hours= 55 min (Fig. 3C), tbest 9 hours= 45 min

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(Fig. 3D) and tbest 12 hours= 42 min (Fig. 3E). Although these tbest are still greater than the

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control at the same sampling time, its recovery from temporary inhibition was obvious. The

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optimum time (tbest) shift every 3 hours is summarized in Fig. 3F in which the stimulation by

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5 mg L-1 Ni2+ was revealed after 6 hours based on the significantly greater ∆tbest of the 5 mg

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L-1 Ni2+ spiked system compared to that of the control.

Figure 3 247

Nickel is not considered as being essential for bacterial growth, although it is found in a

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single hydrogenase of methanogenic bacteria.30 Ni was discovered to be an essential

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component of a low molecular weight coenzyme, F430, present in methanogenic bacteria both

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in the free state and bound to methyl-coenzyme-M reductase, which uses the methyl thioether

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methyl-coenzyme M and the thiol coenzyme B as substrates and converts them reversibly to

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methane in all methanogens.31 At present, F430 has only been found in methanogenic

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archaea.32 Cobalt and other trace metals/elements such as iron, molybdenum, selenium and

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tungsten have also been shown to be stimulatory to methanogens;10 however, it appears that

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nickel is essential and that no other trace metals can replace it in F430.33,34 Besides playing a


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key role in factor F430, nickel is also essential for the growth of acetoclastic organisms; both

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methane producers and sulfate reducing bacteria.7 Depending on the type of substrate, the

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stimulatory and inhibitory concentrations of nickel to anaerobic digestion are between 0.03

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and 27 mg L-1, 35 and 1600 mg L-1 (50% inhibition), respectively.5

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With a Mixture of Co2+ and Ni2+- Because a variety of trace metals are usually present as a

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mixture in the influent of an anaerobic digester, in addition to stimulation, specific

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antagonistic or synergistic effects between mixtures of trace metals can occur in anaerobic

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bioprocesses, and nickel has been proved to be synergistic in the Ni–Co system, i.e.,

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enhancing the toxic/inhibitory effect.35 In the present study of a 2.5+2.5 mg L-1 Ni2+-Co2+

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mixture spiked into the anaerobic system using the resazurin reduction assay, both the

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stimulatory and synergistic effects were demonstrated. In the first 3 hours, compared to the 5

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mg L-1 Co2+ and Ni2+ spiked systems whose tbest was expected to be 75 minutes (Fig. 2B) and

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83 minutes (Fig. 3B), respectively, the activity of the 2.5+2.5 mg L-1 Ni2+-Co2+ spiked system

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was much lower, with a tbest around 130 minutes (Fig. 4B), which did not change significantly

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from the start of the experiment (time 0). This observation suggested that during the first 3

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hours in Ni2+-Co2+ mixture experiment, the addition of Ni2+ greatly enhanced the temporary

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inhibition, which was not observed by measuring methane production in Fig. 1. However,

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after another 3 hours (Fig. 4C), its tbest shifted to 40 minutes, resulting in a ∆tbest 3~6 hours = 90

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minutes (Fig. 4F), which was much greater than the ∆tbest 3~6 hours of the 5 mg L-1 Co2+ and Ni2+

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spiked systems of 37 minutes (Fig. 2F) and 28 minutes (Fig. 3F), respectively, suggesting that

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the speed of recovery from temporary inhibition in the 2.5+2.5 mg L-1 Ni2+-Co2+ spiked

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system was faster than in the single Co2+ (5 mg L-1) or Ni2+ (5 mg L-1) spiked systems.

Figure 4 278

Transition metals nickel and cobalt are essential components of many metalloenzymes; Ni-


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dependent enzymes are urease, [NiFe] hydrogenase, [Ni] superoxide dismutase, CO

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dehydrogenase, and methyl-CoM reductase, while in the form of coenzyme B12, cobalt plays a

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crucial role in many biological functions.15 The synthesis of Ni/Co enzymes, and coenzyme

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B12, requires a high-affinity uptake of these metal ions from natural environments where they

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are available only in trace amounts, and this uptake in bacteria is mediated by various

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secondary transporters and by different ATP-binding cassette systems.15,36 Kida et al. 27

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suggested that during methanogenesis, Ni2+ and Co2+ were required for the methane-

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producing reactions via increases in coenzymes F430 and the corrinoids. In contrast, the toxic

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effect of trace metals is attributed to the disruption of enzyme function and structure by the

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metals binding with thiol and other groups on protein molecules, or by replacing naturally

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occurring metals in enzyme prosthetic groups.35 Therefore, trace metals may be stimulatory or

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inhibitory to anaerobic reactions, and the extent of these effects depends on the metal species

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and its concentration.

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The temporary inhibition of methane production in anaerobic digestion by Ni has been

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reported by Muñoz et al.,37 who proposed that lower nickel concentrations provoke temporary

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inhibition depending on the nickel concentration in the digester. In this present study, the

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same temporary inhibition by Co, Ni, and their mixture at 5 mg L-1, was also observed. Using

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magnetic resonance imaging (MRI), Bartacek et al. (2012) investigated the transport of Co

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inside single methanogenic granules, and found that free Co species tend to interact with the

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granular matrix, and a “reactive barrier” was formed resulting in slower transport (tens of

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minutes to hours). The formation of this reactive barrier seemed to be due to the rate-limiting

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step of transporting metals through the membrane by different transporters,15,36 resulting in

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temporary inhibition. In view of the apparently universal occurrence of F430 in methanogens,

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and corrinoids in methylotrophic methanogens and acetogens, the rate of uptake of free Ni/Co

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followed by the rate of cofactor F430 and corrinoids synthesis, can significant affect the


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activity of the biomass and the amount and rate of methane production. The whole process

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can be described as: (1) Co or Ni diffuse through the biomass and then transfer through the

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cell membrane; (2) Co or Ni join coenzyme synthesis, e.g., cofactor F430 and corrinoids; (3)

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the synthesized coenzymes use specific substrates and convert them to methane. In this

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present study, the resazurin reduction assay was used to determine the bioactivity change in

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anaerobic systems in step 2, while the methane production results measured using the

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volumetric serum bottle method can only be obtained during/after step 3. That is why the

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stimulation of anaerobic digestion by Co/Ni can be assessed using the resazurin reduction

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assay quicker than by measuring methane production.

313

The rate of growth of anaerobic bacteria depends on the type of substrate used. During

314

hydrolysis, 5-72 hours were proposed for carbohydrate and lipid substrates; whereas in the

315

acetogenesis phase, the generation time for bacteria using propionic and fatty acids was

316

suggested to be 84 and 131 hours, respectively; while in the methanogenesis phase,

317

generation times range between 15 and 85 hours.39 During anaerobic digestion, it might take

318

days from a single substrate molecule, for example the peptone or beef extract in our feed, for

319

methane to be produced, while by spiking essential metals, such as Ni, at the stimulatory

320

level, the cofactor F430 will be synthesized and reserved for future use in methanogenesis. To

321

study the effect of Ni and Co on anaerobic digestion, some extracted cofactor F430 and

322

corrinoids were analyzed by atomic absorption spectrophotometer.27 In addition to the

323

drawback of the time-required, this method did not consider the possible effects of increasing

324

F430 and corrinoids on the other metabolic processes in living microbes. In contrast, as a

325

“gross” measure, the resazurin reduction assay can describe the overall activity of mixed

326

cultures, but not a single metabolite. In addition, comparing the activity measured using the

327

present assay and the traditional volumetric method (BMP/ATA), besides the advantage of an

328

earlier and more precision estimate using the resazurin assay, its other advantage is that it is


15


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329

not necessary to purchase expensive equipment such as a GC-TCD to measure methane

330

production to evaluate the activity of anaerobic sludge, while a cheaper fluorescence meter is

331

all that is required for the resazurin assay.

332

Determining the Optimal Concentration of Ni on Stimulation of Anaerobic Digestion by

333

the Resazurin Reduction Assay. By studying the shift in the optimum time of resazurin

334

reduction assay (∆tbest) between two sampling points, the stimulatory effect by metals can be

335

predicted as described above, while the optimal metal concentration at which stimulatory

336

effect is maximized, is still unknown but of practical importance. When studying the

337

mechanism of nickel sorption onto anaerobic granular sludge, Bartacek et al.40 suggested that

338

this dynamic process consisted of three distinctive phases: (1) a fast initial adsorption in the

339

first few minutes after nickel injection, (2) a slow sorption process limited by intra-particle

340

diffusion, and (3) final achievement of equilibrium. Although the second step in the bio-

341

uptake of metals such as nickel (resulting in temporary/permanent toxicity/inhibition) is rate-

342

limiting, e.g., nickel transport into Methanobacterium bryantii cells occurred maximally 4

343

hours after the nickel was spiked;41 the bio-uptake of metals takes place even prior to the start

344

of methanogenic activity, which is much slower than the nickel uptake (several days).

345

Therefore, it is possible for us to estimate methanogenic activity by investigating the

346

distribution of metals in anaerobic sludge.

Figure 5 347

In this present study, by studying the distribution of Ni and Co in anaerobic sludge (Fig. 5),

348

we found that in all the experiments with different spiked metal concentrations, the

349

distribution rate was very fast in the first 6 hours, but then slowed down to almost reach

350

equilibrium in 24 hours. Hence, in this case, we suggest using the 6th hour samples for the

351

resazurin reduction assay to study the effect of different Ni concentrations on anaerobic


16

Page 17 of 31


352

digestion. For anaerobic bioprocesses spiked with a series of Ni2+ concentrations, sludge

353

samples were collected at 6 hours, and their reduction kinetics to resazurin were studied. By

354

plotting the tbest against spiked Ni2+ concentrations (Fig. 6), two critical concentrations can be

355

determined, i.e. stimulation starting (around 1 mg L-1) and stimulation ending (around 100 mg

356

L-1), suggesting that anaerobic digestion can recover from temporary inhibition due to less

357

than 100 mg L-1 of Ni2+ being spiked. Meanwhile, by plotting the slopes of the tbest–Ni2+ curve

358

against metal concentration, the optimal Ni2+ concentration was determined to be around 10

359

mg L-1 (insert Fig. 6). These results were similar to the findings of lower nickel concentrations

360

resulting in reversible inhibition to anaerobic digestion.42

Figure 6 361

Using the present method, four zones for anaerobic digestion with essential trace metal

362

supplementation can be determined; no stimulation, stimulation increase, stimulation decrease

363

and permanent inhibition (Fig. 6). Together with the shift in the optimum time (∆tbest) method,

364

generally within 6 hours using the resazurin reduction assay, the stimulatory effect of

365

essential trace metals on anaerobic digestion can be assessed, and the dose of essential metals

366

on anaerobic process performance can be profiled. By comparing these results to the

367

traditional volumetric method, i.e. measuring methane production over time, we proposed the

368

resazurin reduction assay as a more efficient method to study the stimulation of anaerobic

369

digestion by essential trace metals, with considerable potential for studying the bioavailability

370

of trace metals. The present study developed a novel and rapid assessment method for

371

inhibition and stimulation of anaerobic digestion by essential trace metals, and quickly

372

determined the optimal trace metals dosing strategies for anaerobic digestion. Supplementing

373

exact amounts of essential trace metals to avoid anaerobic digester upset or failure is

374

important, for example, in a rapid response to mitigating temporary inhibition caused by other

375

inhibitors, such as organic toxicants. Future work involves developing a protocol to quantify


17


Page 18 of 31

376

the absolute bioavailability of trace metals in anaerobic digestion based on the resazurin assay,

377

and using this assay in a microfluidic device to fabricate a biosensor for the real-time

378

monitoring of anaerobic digestion metabolic activity.

379 380

AUTHORS INFORMATION

381

Corresponding Authors

382

*phone: +44 207 5945591, fax: +44 15 317453; e-mail: [email protected] (David C.

383

Stuckey); phone: +65-6592-7594, fax: +65-6790-9081; e-mail: [email protected] (Terry

384

W.J. Steele).

385 386

ACKNOWLEDGMENTS

387

This research work was supported by the Singapore National Research Foundation under its

388

Environmental & Water Technologies Strategic Research Programme and administered by the

389

Environment & Water Industry Programme Office (EWI) of the PUB.


18

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390

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Altaş, L. Inhibitory effect of heavy metals on methane-producing anaerobic granular sludge. J. Hazard. Mater. 2009, 162 (2–3), 1551–1556.

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(3)

Gikas, P. Kinetic responses of activated sludge to individual and joint nickel (Ni(II)) and cobalt (Co(II)): An isobolographic approach. J. Hazard. Mater. 2007, 143 (1–2), 246–256.

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Zitomer, D. H.; Johnson, C. C.; Speece, R. E. Metal stimulation and municipal digester thermophilic/mesophilic activity. J. Environ. Eng. 2008, 134 (1), 42–47.

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Lo, H. M.; Chiang, C. F.; Tsao, H. C.; Pai, T. Y.; Liu, M. H.; Kurniawan, T. A.; Chao, K. P.; Liou, C. T.; Lin, K. C.; Chang, C. Y.; et al. Effects of spiked metals on the MSW anaerobic digestion. Waste Manag. Res. 2012, 30 (1), 32–48.

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Fermoso, F. G.; Bartacek, J.; Lens, P. N. L. Effect of vitamin B12 pulse addition on the performance of cobalt deprived anaerobic granular sludge bioreactors. Bioresour. Technol. 2010, 101 (14), 5201–5205.

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Oleszkiewicz, J. A.; Sharma, V. K. Stimulation and inhibition of anaerobic processes by heavy metals: A review. Biol. Wastes 1990, 31 (1), 45–67.

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Chen, Y.; Cheng, J. J.; Creamer, K. S. Inhibition of anaerobic digestion process: A review. Bioresour. Technol. 2008, 99 (10), 4044–4064.

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Chen, J. L.; Ortiz, R.; Xiao, Y.; Steele, T. W. J.; Stuckey, D. C. Rapid fluorescencebased measurement of toxicity in anaerobic digestion. Water Res. 2015, 75 (0), 123– 130.

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Thanh, P. M.; Ketheesan, B.; Yan, Z.; Stuckey, D. Trace metal speciation and bioavailability in anaerobic digestion: A review. Biotechnol. Adv. 2015, 34 (2), 122– 136.

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Nies, D. H. Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid 1992, 27 (1), 17–28.

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Liesegang, H.; Lemke, K.; Siddiqui, R. A.; Schlegel, H. G. Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34. J. Bacteriol. 1993, 175 (3), 767–778.

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Grass, G.; Gro e, C.; Nies, D. H. Regulation of the cnr cobalt and nickel resistance determinant from Ralstonia sp. strain CH34. J. Bacteriol. 2000, 182 (5), 1390–1398.

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Degen, O.; Eitinger, T. Substrate specificity of nickel/cobalt permeases: Insights from mutants altered in transmembrane domains I and II. J. Bacteriol. 2002, 184 (13), 3569– 3577.

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Mulrooney, S. B.; Hausinger, R. P. Nickel uptake and utilization by microorganisms. FEMS Microbiol. Rev. 2003, 27 (2-3), 239–261.

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Battersby, A. R. Biosynthesis of vitamin B12. Acc. Chem. Res. 1993, 26 (1), 15–21.

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Kobayashi, M.; Shimizu, S. Cobalt proteins. Eur. J. Biochem. 1999, 261 (1), 1–9.


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Jefferson, B.; Burgess, J. E.; Pichon, A.; Harkness, J.; Judd, S. J. Nutrient addition to enhance biological treatment of greywater. Water Res. 2001, 35 (11), 2702–2710.

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Mudhoo, A.; Kumar, S. Effects of heavy metals as stress factors on anaerobic digestion processes and biogas production from biomass. Int. J. Environ. Sci. Technol. 2013, 10 (6), 1383–1398.

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Pratten, M.; Ahir, B. K.; Smith-Hurst, H.; Memon, S.; Mutch, P.; Cumberland, P. Primary cell and micromass culture in assessing developmental toxicity. Methods Mol. Biol. 2012, 889, 115–146.

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Chen, J. L.; Steele, T. W. J.; Stuckey, D. C. Modeling and application of a rapid fluorescence-based assay for biotoxicity in anaerobic digestion. Environ. Sci. Technol. 2015, 49 (22), 13463–13471.

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OECD. Test No. 303: Simulation Test - Aerobic Sewage Treatment -- A: Activated Sludge Units; B: Biofilms. OECD Publishing.

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Owen, W. F.; Stuckey, D. C.; Healy Jr, J. B. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 1979, 13 (6), 485–492.

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Lin, C. Y.; Chen, C. C. Effect of heavy metals on the methanogenic UASB granule. Water Res. 1999, 33 (2), 409–416.

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Rozzi, A.; Remigi, E. Methods of assessing microbial activity and inhibition under anaerobic conditions: A literature review. Rev. Environ. Sci. Biotechnol. 2004, 3 (2), 93–115.

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Sanders, W. T.; Zeeman, G.; Lettinga, G. Hydrolysis kinetics of dissolved polymer substrates. Water Sci. Technol. 2002, 45 (10), 99–104.

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Kida, K.; Shigematsu, T.; Kijima, J.; Numaguchi, M.; Mochinaga, Y.; Abe, N.; Morimura, S. Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis. J. Biosci. Bioeng. 2001, 91 (6), 590–595.

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Florencio, L.; Field, J. A.; Lettinga, G. Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium. Appl. Environ. Microbiol. 1994, 60 (1), 227–234.

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Krzycki, J.; Zeikus, J. G. Quantification of corrinoids in methanogenic bacteria. Curr. Microbiol. 1980, 3 (4), 243–245.

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Jarrell, K. F.; Kalmokoff, M. L. Nutritional requirements of the methanogenic archaebacteria. Can. J. Microbiol. 1988, 34 (5), 557–576.

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Friedmann, H. C.; Klein, A.; Thauer, R. K. Biosynthesis of Tetrapyrroles; New Comprehensive Biochemistry; Elsevier, 1991; Vol. 19.

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Thauer, R. K. Biochemistry of methanogenesis: A tribute to Marjory Stephenson. In Microbiology; 1998; Vol. 144, pp 2377–2406.

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Speece, R. E.; Parkin, G. F.; Gallagher, D. Nickel stimulation of anaerobic digestion. Water Res. 1983, 17 (6), 677–683.

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(34)

Zhang, P.; Shen, Y.; Guo, J.-S.; Li, C.; Wang, H.; Chen, Y.-P.; Yan, P.; Yang, J.-X.; Fang, F. Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics. Sci. Rep. 2015, 5 (January), 12041.

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Eitinger, T.; Suhr, J.; Moore, L.; Smith, J. A. C. Secondary transporters for nickel and cobalt ions: theme and variations. Biometals 2005, 18 (4), 399–405.

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Muñoz, M. A.; Codina, J. C.; De Vicente, A.; Sanchez, J. M.; Borrego, J. J.; Moriñigo, M. A. Effects of nickel and lead and a support material on the methanogenesis from sewage sludge. Lett. Appl. Microbiol. 1996, 23 (5), 339–342.

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Bartacek, J.; Fermoso, F. G.; Vergeldt, F.; Gerkema, E.; Maca, J.; van As, H.; Lens, P. N. L. The impact of metal transport processes on bioavailability of free and complex metal ions in methanogenic granular sludge. Water Sci. Technol. 2012, 65 (10), 1875– 1881.

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Ali Shah, F.; Mahmood, Q.; Maroof Shah, M.; Pervez, A.; Ahmad Asad, S. Microbial ecology of anaerobic digesters: the key players of anaerobiosis. Sci. World J. 2014, 2014, 183752.

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(40)

Bartacek, J.; Fermoso, F. G.; Catena, A. B.; Lens, P. N. L. Effect of sorption kinetics on nickel toxicity in methanogenic granular sludge. J. Hazard. Mater. 2010, 180 (1-3), 289–296.

489 490

(41)

Jarreli, K. F.; Sprott, G. D. Nickel transport in Methanobacterium bryantii. J. Bacteriol. 1982, 151 (3), 1195–1203.

491 492

(42)

Mueller, R. F.; Steiner, A. Inhibition of anaerobic digestion caused by heavy metals. In Water Science and Technology; 1992; Vol. 26, pp 835–846.

493


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494

Figure and Scheme Captions:

495

Scheme 1. Conversion of resazurin to resorufin and dihydroresorufin.21

496

At a λex of 530 nm and λem of 590 nm, resazurin is blue and weakly fluorescent; resorufin is

497

pink and strongly fluorescent; dihydroresorufin is colorless and non-fluorescent.

498

Fig. 1. Effects of spiked Ni2+, Co2+ and their mixture on methane production.

499

The volume of methane produced with Ni2+ (a), Co2+ (b) and their mixture (c) spiked was

500

measured every 3 hours in the first 12 hours (A), and daily (B). Mean and standard deviation

501

of three replicates are shown. The superscript * indicates there is a significant difference

502

between these two groups of data according to a t-test at P ≤ 0.05.

503

Fig. 2. Resazurin reduction assay to study stimulation of anaerobic process by Co.

504

Resazurin reduction by anaerobic sludge sampled at 0 (A), 3 (B), 6 (C), 9 (D) and 12 (E)

505

hours with 0, 5 and 250 mg L-1 Co2+ spiked, and the shift of optimum time for maximizing

506

produced resorufin in a fluorescent signal (tbest) every 3 hours was summarized (F), where the

507

superscript * indicates there is a significant difference between these two groups of data

508

according to a t-test at P ≤ 0.05.

509

Fig. 3. Resazurin reduction assay to study stimulation of anaerobic process by Ni.

510


511

hours with 0, 5 and 250 mg L-1 Ni2+ spiked, and the shift of optimum time for maximizing

512


513


514


515

Fig. 4. Resazurin reduction assay to study stimulation of anaerobic process by mixture

516

of Co and Ni.

517


518

hours with 0, 2.5-2.5 mg L-1 Ni2+-Co2+ spiked, and the shift of optimum time for maximizing

519


520


521



22

Page 23 of 31


522

Fig. 5. Dynamic distribution of Ni2+, Co2+ and their mixture in the liquid phase of

523

anaerobic process.

524

The initial metals concentration is: Co2+ at 5 (A) and 250 (B) mg L-1; Ni2+ at 5 (C) and 250 (D)

525

mg L-1; and mixture of Ni2+-Co2+ at 2.5-2.5 mg L-1 (E). Mean and standard deviation of three

526

replicates are shown.

527

Fig. 6. Effect of Ni2+ dose on anaerobic process performance studied by optimum time

528

for maximizing produced resorufin in resazurin Reduction assay.

529

Mean and standard deviation of three replicates are shown. By plotting the slopes of tbest–Ni2+

530

curve against metal concentration (insert Figure), the optimal Ni2+ concentration was

531

determined to maximize the methane production rate in anaerobic bioprocess.


23


Page 24 of 31

532 533

Scheme 1


24

Page 25 of 31


A

CH4 (mL)

0.3

Control Ni2+ 5 mg L-1 2+ -1 Ni 250 mg L

0.4

0.3

a

0.2

0.1

0.2

0.4

*

0.1

0.0 6

9

12

0.2

c *

0.1

0.0 3

Control Co2++Ni2+ 2.5+2.5 mg L-1

0.3

b

CH4 (mL)

Control Co2+ 5 mg L-1 Co2+ 250 mg L-1

CH4 (mL)

0.4

0.0 3

6

9

12

3

6

Time (hour)

Time (hour)

9

12

Time (hour)

B Control Co2+ 5 mg L-1 Co2+ 250 mg L-1

12

*

a 6

3

0

12

*

9

*

CH4 (mL)

CH4 (mL)

9

*

Control Ni2+ 5 mg L-1 Ni2+ 250 mg L-1

b 6

* 3

2

Time (day)

3

*

*

c 6

* 3

0 1

Control 2+ 2+ -1 Co +Ni 2.5+2.5 mg L

9

CH4 (mL)

12

0 1

2

Time (day)

3

1

2

3

Time (day)

Fig. 1

25



A

B 0 hour

200

fluorescence (RFU)

fluorescence (RFU)

200

3 hours

150

150

100

100

50

50

0

0

0

10

20

30

40

50

60

0

10

TIME (min)

C

D

30

40

50

60

6 hours

50

60

200

fluorescence (RFU)

fluorescence (RFU)

200

150

9 hours

150

100

100

50

50

0 0

10

20

30

40

50

60

0

10

20

30

40

TIME (min)

TIME (min)

F

200

fluorescence (RFU)

20

TIME (min)

0

12 hours

*

Control Co2+ 5 mg L-1

90

150

∆tbest (min)

E

Page 26 of 31

100

50

60

* 30

* * 0

0 0

10

20

30

40

50

60

0~3 hours 3~6 hours 6~9 hours 9~12 hours

TIME (min)

Fig. 2

26 ACS Paragon Plus Environment

Page 27 of 31

B

200

0 hour

fluorescence (RFU)

fluorescence (RFU)

A


150

100

50

200

3 hours 150

100

50

0

0

0

10

20

30

40

50

0

60

10

TIME (min)

D

200

6 hours 150

100

50

50

60

50

60

9 hours 150

100

50

0 0

10

20

30

40

50

60

0

10

TIME (min)

20

30

40

TIME (min)

F

200

12 hours

*

Control Ni2+ 5 mg L-1

90

150

∆tbest (min)

fluorescence (RFU)

40

200

0

E

30

TIME (min)

fluorescence (RFU)

fluorescence (RFU)

C

20

100

50

60

* 30

* *

0 0

10

20

30

40

50

0

60


TIME (min)

Fig. 3 27 ACS Paragon Plus Environment


B

200

200

fluorescence (RFU)

fluorescence (RFU)

A

0 hour 150

100

50

3 hours 150

100

50

0

0

0

10

20

30

40

50

60

0

10

TIME (min)

D

200

6 hours 150

100

50

40

50

60

50

60

9 hours 150

100

50

0 0

10

20

30

40

50

60

0

10

TIME (min)

20

30

40

TIME (min)

F

200

12 hours

*

Control

*

90

∆tbest (min)

fluorescence (RFU)

30

200

0

E

20

TIME (min)

fluorescence (RFU)

fluorescence (RFU)

C

Page 28 of 31

150

100

50

Co2+ + Ni2+ 2.5+2.5 mg L-1

60

30

* *

0

0 0

10

20

30

40

50

60


TIME (min)

Fig. 4


Page 29 of 31


B 1.5

Co2+ in liquid (mg L-1)

Co2+ in liquid (mg L-1)

A Co 5 mg L-1 spiked

1.0

0.5

0.0

240

Co 250 mg L-1 spiked

200

160

120 0

5

10

15

20

25

0

5

Time (hour)

15

20

25

Time (hour)

D

4

240 Ni 5 mg L-1 spiked

Ni2+ in liquid (mg L-1)

Ni2+ in liquid (mg L-1)

C

10

3

2

1

Ni 250 mg L-1 spiked 200

160

120

0 0

5

10

15

20

25

0

5

10

15

20

25

Time (hour)

Time (hour)

Metal in liquid (mg L-1)

E 2.0

Ni 2.5 mg L-1 spiked Co 2.5 mg L-1 spiked

1.5 1.0 0.5 0.0 0

5

10

15

20

25

Time (hour)

Fig. 5



Page 30 of 31

Fig. 6


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