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CHEMICAL ENGINEERING TRANSACTIONS

VOL. 49, 2016

A publication of

The Italian Association

of Chemical Engineering
Online at www.aidic.it/cet

Guest Editors: Enrico Bardone, Marco Bravi, Tajalli Keshavarz
Copyright © 2016, AIDIC Servizi S.r.l.,
ISBN 978-88-95608-40-2; ISSN 2283-9216

Impact of Magnetite Nanoparticles Supplementation on the
Anaerobic Digestion of Food Wastes: Batch and Continuous-

Flow Investigations

Claudia Dalla Vecchiaa, Andrea Mattiolia, David Bolzonellaa*, Enza Palmab,
Carolina Cruz Viggib, Federico Aulenta b.
a
Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

b
Water Research Institute (IRSA), National Research Council (CNR), via Salaria km 29.300, 00015 Monterotondo (RM),

david.bolzonella@univr.it

Anaerobic digestion is one of the most attractive technologies for the treatment of industrial, civil and
agricultural organic waste because of its capacity to reduce the biodegradable matter while recovering
renewable energy in the form of methane. However, this bioprocess sometime suffers of problems in the
interspecies electron transfer between acetogenic bacteria and methanogenic archaea with consequent yields
reduction or failure. Recently, direct interspecies electron transfer between species via solid conductive
materials like nanoparticles has been observed. This study, in particular, examined the effect of magnetite
nanoparticles supplementation on the methanogic conversion of organic substrates both in batch trials using
pure compounds (i.e., propionate and butyrate) and continuous trials using real food waste as substrate..
Batch experiments demonstrated once again the validity of the proposed approach, whereby the conductive
particles likely promoted the occurrence of direct interspecies electron transfer processes between acetogens
and methanogens. Notably, continuous experiments confirmed the significance of this mechanism also for the
treatment of real substrates, although the relative magnitude of the stimulatory effect was slightly lower.

1. Introduction
Anaerobic digestion is one of the most attractive technologies for the treatment of industrial wastewaters and
organic wastes, because it requires less energy investments and generates less excess sludge than other
treatment approaches such as the activated sludge system (Mata-Alvarez et al., 2000).
Complete conversion of organic matter to CO2 and CH4 via anaerobic digestion typically requires the
syntrophic cooperation between acetogenic bacteria (also referred to as syntrophs) and methanogenic
archaea. Indeed, catabolic reactions catalysed by acetogenic bacteria become energetically favourable only
when produced reducing equivalents are efficiently scavenged by their syntrophic partners, namely the
methanogenic archaea. Typically, this interspecies electron transfer (IET) process is reported to occur via
diffusive transport of soluble electron carriers (e.g., hydrogen and formate) from the acetogens to the
methanogens (Morris et al., 2013; Stams and Plugge, 2009). Low concentrations of electron carriers however
result in slow diffusion rates, causing IET to be often the bottleneck in the methanogenic conversion of organic
substrates. Recently, direct interspecies electron transfer (DIET), in which two microbial species exchange
electrons via electric currents flowing through conductive solid conduits (e.g., magnetite nanoparticles) or
microbial pili, has been proposed as an alternative strategy to interspecies H2/formate transfer, through which
microbial species in a community share reducing equivalents to drive the methanogenic degradation of
organic substartes (Kouzuma et al., 2015; Shrestha and Rotaru, 2014).
Numerous studies reveal that DIET could be sustained and stimulated by the addition of electrically
conductive materials, including granulated activated carbon (Liu et al., 2012), biochar (Chen et al., 2014), and
naturally occurring iron minerals (hematite or magnetite), of nanometer to micrometer size (Zhuang et al.,
2015; Cruz Viggi et al., 2014; Li et al., 2014; Zhou et al., 2014; Kato et al., 2012). These electrically conductive

DOI: 10.3303/CET1649001

Please cite this article as: Dalla Vecchia C., Mattioli A., Bolzonella D., Palma E., Cruz Viggi C., Aulenta F., 2016, Impact of magnetite
nanoparticles supplementation on the anaerobic digestion of food wastes: batch and continuous-flow investigations, Chemical Engineering
Transactions, 49, 1-6 DOI: 10.3303/CET1649001

materials function as electron conduits for direct electron transfer between syntrophic, organic matter-oxidizing
bacteria and CO2-reducing methanogens.
So far, however, this interesting mechanism has been only observed in short-term batch experiments in the
presence of synthetic substrates of defined composition, whereas its relevance and practical viability under
conditions more closely resembling those occurring in real anaerobic digesters remains largely unknown.
In order to address this issue, in the present study we investigated the impact and practical feasibility of
magnetite nanoparticles supplementation on the anaerobic digestion of “real” food wastes. To this aim, both
batch and continuous-flow mesophilic anaerobic digestion experiments with or without magnetite
supplementation using mixed microbial cultures were carried out.
Firstly, the influence of magnetite particles onto anaerobic digestion was assessed setting up a series of batch
tests using an unacclimated methanogenic sludge as inoculum and proprionate and butyrate, chosen as
model substrates to study energy-limited syntrophic communities. Then, a continuous study was set up to
verify the influence of magnetite supplementation on anaerobic digestion process in stirred reactors where real
food waste was the feed.

2. Materials and methods
2.1 Batch experiments with model substrates

Magnetite nanoparticles were synthesized according to a previously described protocol (Cruz Viggi et a.,
2014). All batch experiments were conducted in anaerobic 120 mL serum bottles incubated statically, in the
dark, at room temperature (20−25 °C). Bottles contained 57 mL of mineral medium [NH4Cl (0.5 g/L),
MgCl2·6H2O (0.1 g/L), K2HPO4 (0.4 g/L), and CaCl2·2H2O (0.05 g/L)], 1 mL of sodium bicarbonate (10
wt%/wt), and either 2 mL of a suspension of magnetite nanoparticles, corresponding to a final concentration of
0.35 g Fe/L (magnetite-amended bottles), or 2 mL of deionized water (unamended controls). Upon
preparation, all bottles were sealed with Teflon-faced butyl rubber stoppers, flushed with a 70% N2/30% CO2
gas mixture, inoculated with 1 mL of anaerobic methanogenic culture [corresponding to an initial volatile
suspended solids (VSS) concentration of 0.20 g/L] and spiked with propionate or butyrate to a final
concentration of approximately 2.5 mmol/L.
Throughout all incubations, the pH remained in the range of 7.5−7.8. During the incubations, the bottles were
sampled for the determination of butyrate, propionate, acetate and methane concentrations.

2.2 Continous experiments

Two bench scale continuously stirred reactors (CSTR) operating in mesophilic (37°C) environment were used
for the anaerobic digestion trials: one reactor was not supplemented with magnetite and was used as control
(blank reactor, B), while the second one was added with magnetite nanoparticles (M reactor) prepared like
described above. The operating volume of the two reactor was 4.5 L while organic loading rate (OLR) and
hydraulic retention time (HRT) were fixed at 1.8 gTVS per litre per day and 15 days, respectively. The
characteristics of the food waste used in the experimentation are are shown in table 1: this was a synthetic
feed due to the mix of meat, fruit and pasta, with a considerable presence of carbohydrates.

Table 1: Feed characteristics

Parameter Average values

Total Solids, g/kg 303

Total Volatile Solids, g/kg 297

COD, g/kg 292

TKN, gN/kg 7.8

2.3 Analytical Methods

Organic acids (acetate, propionate and butyrate) were analyzed by injecting 1 μL of filtered (0.22 μm porosity)
liquid sample into a PerkinElmer Auto System gas chromatograph (2 m × 2 mm stainless steel column packed

with phase 0.3% Carbowax 20M + 0.1% H3PO4, matrix 60/80 Carbopack C support, Supelco; N2 carrier gas
20 mL/min; oven temperature 120 °C; injector temperature 200 °C; flame ionization detector (FID)
temperature 200 °C). Methane was analyzed by injecting 50 μL of headspace sample (with a gastight
Hamilton syringe) into a Perkin-Elmer Auto System gas chromatograph (stationary phase: stainless-steel
column packed with molecular sieve (Supelco, USA); carrier gas: N2 at 20 mL/min; oven temperature: 150 °C;
injector temperature: 200 °C; thermal conductivity detector (TCD) temperature: 200 °C).
Analysis of pH, total solids (TS), total volatile solids (TVS), chemical oxygen demand (COD), ammonia, total
Khjeldahl nitrogen (TKN), and partial and total alkalinity were all carried out according to the Standard
Methods for Water and Wastewater Analysis recommendations. Volatile fatty acids (VFA) from C2 (acetate)
to C5 (valerate) and their normal and isoforms in the continuous anaerobic reactors were carried out using
liquid chromatography as described in Raposo et al (2015).

3. Results and discussion
3.1 Batch experiments with model substrates

In order to preliminary assess the influence of magnetite nanoparticles on the kinetics of anaerobic digestion,
a series of batch tests were conducted using an unacclimated methanogenic sludge as inoculum and
propionate and butyrate as model substrates. In these experiments, propionate and butyrate degradation, in
bottles containing magnetite nanoparticles, started after a slightly shorter lag phase and proceeded to
completion more rapidly compared to the unamended control bottles.
Apparently, the pathway of propionate and butyrate degradation was not affected by the presence of
magnetite; both in magnetite-supplemented and control bottles the methanogenic conversion of butyrate
proceeded via the intermediate formation of propionate and acetate (and likely hydrogen although this latter
compounds was not determined), whereas the methanogenic conversion of propionate proceed via the
intermediate formation of acetate (and likely hydrogen). In spite of that, however, in magnetite-supplemented
bottles the rate of substrates degradation and methane formation were higher than in the corresponding
controls (Figure 1). Specifically, in magnetite-supplemented bottles the maximum rate of methane formation
(on a molar basis) from propionate and butyrate were respectively 22% and 12% higher than in the
corresponding unamended controls (Figure 1a). In agreement with that, the rates of propionate and butyrate
degradation were correspondingly higher (Figure 1b).

Figure 1: Effect of magnetite nanoparticles supplementation on (a) the maximum rate of methane formation
and (b) the maximum rate of propionate or butyrate degradation.

Collectively, these results are in good agreement with those reported in a previous work (Cruz Viggi et al.,
2014), in which we demonstrated that supplementation of micrometer-size magnetite particles to a
methanogenic sludge enhanced (up to 33%) the rate of methane generation from propionate.
Taken as a whole, these findings suggest that the stimulatory effect of electrically conductive nanoparticles
results from the establishment of a direct interspecies electron transfer (DIET) process, based on magnetite
particles serving as electron conduits between propionate- or butyrate-oxidizing acetogens and carbon
dioxide-reducing methanogens. This novel IET mechanism, schematically depicted in Figure 2, could allow
overcoming some of the kinetic limitations of traditional, diffusion-based, interspecies H2 transfer (Figure 2). .

Figure 2: Proposed electron transfer mechanisms between an acetogen and a methanogen in magnetite
supplemented cultures: interspecies H2 transfer (A) and electronic conduction through magnetite particles (B).

3.2 Continous experiments

Two different series of continuous trials were carried out: in the first one, the two reactors operated as
continuous reactors. In these conditions the reactors operated for few HRTs and then failed because of
volatile fatty accumulation. In a second series of experiments part of the solids effluent were recycled into the
system so to uncouple the hydraulic and solids retention time and improve the available active biomass and
buffer capacity of the system: this strategy demonstrated the possibility to operate the two continuous reactors
for an indefinite time.
With specific reference to the experimental results, in the first run, without biomass recycling, the magnetite
was added in the feed daily at a load of 10 mmol per day. The supplemented reactor showed higher
performances in terms of biogas production but also an higher stability in typical process parameters like pH,
VFA and partial and total alkalinity (data not shown).
On the other hand, the control reactor (B) operated steadily for one HRT and after that time (15 days) VFA
accumulated in the systems reaching concentrations of 10-12 grams per litre. Propionic acid was the main
compound found in the mixture of organic acids accumulated in the reactor.
Overall, comparing the performances of the two reactors it turned out that the supplemented reactor was more
stable and was able to operate for an additional HRT with respect to the control reactor. Also biogas biogas
production was slightly higher. On the other hand, surprisingly, the control reactor showed an higher methane
concentration in biogas (59 vs. 56%). Table 1 resumes the main findings of the continuos operation trials.
In the second series of experiments the recirculation strategy was introduced to confer robustness to the
process: in particular, the effluent of the two anaerobic digesters, both the control and the supplemented one,
was centrifuged and 50% of the solid pellet was recirculated into the reactors. Clearly, in this way, also part of
the magnetite was recirculated into the reactor. In order to maintain a magnetite concentration in the
supplemented reactor similar to the one observed in the first series of experiments the magnetite load was
lowered to 3.3 mmol per day. This load was determined on the basis of the system mass balance.
As a consequence of the partial recirculation of anaerobic biomass the systems showed stable parameters
and VFA concentrations that never overpassed a level of 200 mgCOD/L. Similarly to the first series of
continuous and batch tests, the supplemented reactor (M) presented higher biogas productions.
In particular, the specific biogas production in the supplemented reactor passed from 0.67 to 0.72 L/gVS with
a 8% increase. Also the biogas production rate showed a similar trend, passing from 2.4 to 2.6 litre biogas per
litre reactor per day. As in the first experimental run the methane percentage was slightly lower (some
percentage points) in the supplemented reactor. Operational applied conditions (OLR and HRT) and yields
(SGP and GPR) for the whole continuous experimentation are shown in table 2.
In general, the continuous experiments confirmed and put in major evidence that effect of the magnetite
nanoparticles addition: in particular, it was emphasised how the addition of magnetic particles allowed for an
increase in the biogas production also when real substrates are used and confirmed the results reported in
similar studies (Hellman et al., 2012).

Propionate
or Butyrate

Acetate

Bicarbonate

Methane

H2 Methanogen
Acetogen

Acetate

Bicarbonate

Methane

Magne te
electron conduit

e- e
-

a b
Propionate
or Butyrate

In general, it should be highlighted that continuous experiments confirmed results obtained in batch
experiments related to the mechanism study but the improvement herein obtained was of lower magnitude, as
expected for continuous reactors when compared to batch ones.

Table 2: Operative conditions without and with solid recirculation of the two digesters

Control (*) Supplemented (*) Control Supplemented

Biomass recycling No No Yes Yes

OLR, gVS/L·d 1.8 1.8 1.8 1.8

HRT, d 15 15 15 15

SRT, d 15 15 32 32

SGP, L/gVS 0.53 ± 0.12 0.66 ± 0.13 0.67 ± 0.01 0.72 ± 0.01

GPR, L/L·d 1.9 ± 0.4 2.3 ± 0.5 2.4 ± 0.3 2.6 ± 0.3

CH4, % 59.2 ± 5.6 56.3 ± 2.9 54.3 ± 1.6 52.8 ± 2.0

(*) System failure at the second HRT

4. Conclusions
This study examined the effect of magnetite nanoparticles supplementation on the methanogic conversion of
organic substrates. To this aim, both batch trials using pure compounds (i.e., propionate and butyrate) and
continuous trials using real food waste as substrate were carried out. Batch experiments demonstrated once
again the validity of the proposed approach, whereby the condutive particles likely promoted the occurrence of
direct interspecies electron transfer processes between acetogens and methanogens. Notably, continuous
experiments confirmed the significance of this mechanism also for the treatment of real substrates, although
the relative magnitude of the stimulatory effect was slightly lower.

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