Journal of Environmental Management 95 (2012) S355eS359

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Re-fermentation of washed spent solids from batch hydrogenogenic fermentation

for additional production of biohydrogen from the organic fraction of municipal
solid waste
Karla M. Muoz-Pez a, Elvira Ros-Leal b, Idania Valdez-Vazquez a, Noem Rinderknecht-Seijas c,
Hctor M. Poggi-Varaldo a, *
a

Environmental Biotechnology and Renewable Energies R&D Group, Dept. Biotechnology and Bioengineering, Centro de Investigacin y de Estudios Avanzados del Instituto
Politcnico Nacional, P.O. Box 14-740, Mxico D.F. 07000, Mexico
Central Analtica, Dept. Biotechnology and Bioengineering, Centro de Investigacin y de Estudios Avanzados del Instituto Politcnico Nacional, Mxico D.F., Mexico
c
ESIQIE del IPN, Divisin de Ciencias Bsicas, Campus Zacatenco, Mxico D.F., Mexico
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 27 September 2009
Received in revised form
16 November 2010
Accepted 18 January 2011
Available online 12 February 2011

In the rst batch solid substrate anaerobic hydrogenogenic fermentation with intermittent venting
(SSAHF-IV) of the organic fraction of municipal solid waste (OFMSW), a cumulative production of
16.6 mmol H2/reactor was obtained. Releases of hydrogen partial pressure rst by intermittent venting
and afterward by ushing headspace of reactors with inert gas N2 allowed for further hydrogen
production in a second to fourth incubation cycle, with no new inoculum nor substrate nor inhibitor
added. After the fourth cycle, no more H2 could be harvested. Interestingly, accumulated hydrogen in 4
cycles was 100% higher than that produced in the rst cycle alone. At the end of incubation, partial
pressure of H2 was near zero whereas high concentrations of organic acids and solvents remained in the
spent solids. So, since approximate mass balances indicated that there was still a moderate amount of
biodegradable matter in the spent solids we hypothesized that the organic metabolites imposed some
kind of inhibition on further fermentation of digestates. Spent solids were washed to eliminate organic
metabolites and they were used in a second SSAHF-IV. Two more cycles of H2 production were obtained,
with a cumulative production of ca. 2.4 mmol H2/mini-reactor. As a conclusion, washing of spent solids of
a previous SSAHF-IV allowed for an increase of hydrogen production by 15% in a second run of SSAHF-IV,
leading to the validation of our hypothesis.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Biohydrogen
Organic fraction of municipal solid wastes
Re-fermentation
Solid substrate
Spent solids
Washing

1. Introduction
In the last 10 years, interest on biohydrogen has resurrected,
particularly the research on dark fermentation of solid wastes
(Kovcs et al., 2006; Muoz-Pez et al., 2008a). Hydrogen can be
considered the best energy alternative because it can be produced
by biological means, it has the highest energy density, it is versatile
since can be used both as a primary or secondary energy source,
and it is environmentally-friendly since water is the main
combustion product and no aggressive pollutants are generated
(Mizuno et al., 2002).
In bioydrogen production by dark fermentation of organic
wastes, several microbial groups that consume H2 coexist with the

* Corresponding author. Tel.: 5255 5747 3800x4324/4321; fax: 5255 5747

3313.
E-mail address: hectorpoggi2001@gmail.com (H.M. Poggi-Varaldo).
0301-4797/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jenvman.2011.01.017

H2-producing microbes (Valdez-Vazquez and Poggi-Varaldo, 2009).

So, it is paramount to process feasibility to nd techniques to
inhibit the H2-consuming microorganisms, such as the methanogenic archaea to cite one of the most important groups. It has been
reported a variety of methanogenesis inhibitors, inter alia: acetylene, bromo-ethanesulphonate (BES), heat-shock pretreatment and
low pH (Lay et al., 1999; Valdez-Vazquez and Poggi-Varaldo, 2009;
Sprott et al., 1982; Sparling et al., 1997). Methanogenesis inhibition
by low pH has been less studied in solid substrate hydrogenogenic
fermentation, although it seems to be attractive and feasible for
practical applications since low pH would be naturally driven by
the accumulation of organic acids in the acidogenic fermentation of
organic matter (Muoz-Pez et al., 2008a). Environmental pH is
a factor that has a variety of effects on the physiology of microorganisms. For instance, pH impacts on the electrical charge of the
cell membranes, which in turn may inuence the uptake of
substrates and nutrients. Also, pH may have a noticeable effect on
enzymatic activities. It is known that pH may determine to a great

S356

K.M. Muoz-Pez et al. / Journal of Environmental Management 95 (2012) S355eS359

extent the type and distribution of fermentation products. In biohydrogen fermentation, the pH strongly inuences the active
bacteria, affecting among others the overall H2 production, the
specic rate of H2 generation, and type and concentration of
organic acids and solvents (Li et al., 2007).
Another important aspect in biohydrogen fermentation is the
possible inhibition of further hydrogen production by accumulation
of nal products. The accumulation of dissolved H2 in the liquid
phase (associated to high partial pressures of hydrogen in the gas
phase) could inhibit hydrogen production (Logan et al., 2002;
Sparling et al., 1997; Valdez-Vazquez et al., 2005) and along with
low pH it may promote the shift to solventogenic fermentation.
Bioreactor headspace release by venting and eventual ushing
with inert gas seems to alleviate such an inhibition (Mizuno et al.,
2000; Valdez-Vazquez et al., 2006a).
There is little information on inhibition of the biohydrogenogenesis by accumulation of organic acids and other organic
metabolites. It is known that undissociated (or unionized) forms of
organic acids are more inhibitory to fermentative bacteria than the
anionic forms. It seems that unionized organic acids may uncouple
the growth of microorganisms (Booth, 1985; Wang and Wang,
1984). It is likely that hydrogen production cessation in batch
solid substrate anaerobic hydrogenogenic fermentation with
intermittent venting and headspace ushing (SSAHF-IV) of organic
fraction of municipal solid wastes (OFMSW) at the end could also
be due to accumulation of organic acids and other organic metabolites (Valdez-Vazquez et al., 2005; van Ginkel and Logan, 2005).
Therefore, the objective of this work was to evaluate the
hydrogenogenic second fermentation of washed spent solids originated in a rst SSAHF-IV. Washing of spent solids was performed
between batch fermentations in order to eliminate the presence of
organic metabolites.
2. Materials and methods
2.1. Experimental design and bioreactors
2.1.1. First SSAHF-IV of organic fraction of municipal solid wastes
The process chosen was the so-called mesophilic (35  C), batch,
solid substrate anaerobic hydrogen fermentation with intermittent
venting and headspace ushing (SSAHF-IV) according to procedures reported by Valdez-Vazquez et al. (2005).
2.1.1.1. Inocula. Mini-reactors were seeded with digestates from
methanogenic solid substrate anaerobic digesters degrading
a mixture of organic solid wastes. Those digesters were operated at
21 days mass retention time in mesophilic conditions.
2.1.1.2. Substrate. The substrate was a model organic fraction of
municipal solid waste (OFMSW) conformed by a mixture of 40% of
paper and 60% food waste. It was prepared with paper and food
wastes from CINVESTAVs cafeteria. The mixture was vacuum-dried
overnight, milled and stored at 58  C until its use. The substrate
moisture content was adjusted to 25% total solids with a buffer
containing 31.6 g NaHCO3 and 63.3 g K2HPO4 per liter; buffer and
substrate were sterilized in order to control the studied microbial
consortium producers of H2. The main characteristics of the feedstock supplied to the mini-reactors are shown in Table 1.
2.1.1.3. Mini-reactors and experimental procedure. Wide mouth
glass bottles of 250 mL volume which were tightly stoppered using
rubber stoppers and metallic harnesses were used as mini-reactors.
First, in an anerobic glove chamber, 20 g of inocula y 80 g of
substrate were added to mini-reactors and blended with the
inocula. Background controls with only 20 g of inoculum and

Table 1
Characterization of substrates in both solid substrate anaerobic hydrogenogenic
fermentation with intermittent venting and headspace ushing.
Parameter

TSb(%)
VSc(%)
TKN (%)d
Cellulose (%)d
Lignin (%)d
Phosphorus (mg/kg)
PH

SSAHF-IVa
1

Organic fraction of municipal

solid wastes

Washed spent solids

24.54
14.90
1.93
26.2
19.4
17.3
7.34









0.13
0.85
0.80
1.5
1.1
1.1
0.15

19.62  0.03
8.91  0.02
NDe
18.7  0.8
16.9  1.2
13.7  0.3
7.0  0.20

a
Notes: batch solid substrate anaerobic hydrogenogenic fermentation with
intermittent venting and ushing of reactor headspace.
b
Total solids.
c
Volatile solids.
d
% of total solids.
e
Not determined.

abiotic controls (tyndalysed substrate plus inoculum) were also

run. Next, mini-reactors were made anerobic under N2 using an
evacuation-gas replacement method. At time zero, all bioreactor
headspaces were injected with acetylene 1% v/v (Sparling et al.,
1997). The mini-reactors were incubated in a constant temperature room at 37  C.
The bioreactors were rst subjected to SSAHF-IV of ve cycles,
with no addition of new inocula and acetylene. During each cycle,
bioreactor headspace was intermittently vented at atmospheric
pressure. At the end of each cycle, reactor headspace was ushed
with N2 in order to decrease hydrogen concentration down to zero.
Once it was determined the absence of hydrogen production in the
fth cycle, the spent solids were washed (1 part of spent solids in 3
parts of tap water), settled, manually squeezed, and dried for future
use.
2.1.2. Second SSAHF-IV of washed, spent solids from the rst
fermentation
The washed spent solids from the rst SSAHF-IV were loaded
along with fresh inocula in bioreactors and subjected to a second
SSAHF-IV with a similar procedure as described above. This time,
inhibition of methanogenesis was effected by setting pH at 6.3 with
a buffer solution (Muoz-Pez et al., 2008b). Bioreactor incubation
and headspace handling were similar to procedures outlined in
Subsection 2.1.1. All the experiments of the rst and second SSAHFIV were run by duplicate.
The main characteristics and properties of the OFMSW used in
the rst SSAHF-IV and the spent solids fed to the second SSAHF-IV
are exhibited in Table 1.
2.2. Analyses
The main monitoring parameters were hydrogen production,
initial and nal pH, and solids contents. Hydrogen and methane
contents in biogas were determined by gas chromatography (PoggiVaraldo et al., 1997) in a GoweMac chromatograph model 350 tted
with a thermal conductivity detector (TCD) and Molecular Sieve 5A
packed column (injector, detector and column temperatures were
25, 100 and 25  C, respectively). Argon was the carrier gas. Volatile
organic acids and solvents were determined in water extracts of the
spent solids of mini-reactors: 5 g of homogenized spent solids was
thoroughly mixed with 25 mL of distilled water. The suspension was
ltered through a glass-membrane lter and an aliquot of the
ltrate was injected in a gas chromatography Varian Star 3400
equipped with a FID for metabolite concentration determination.

K.M. Muoz-Pez et al. / Journal of Environmental Management 95 (2012) S355eS359

The injector and detector temperatures were set at 250  C. Nitrogen

was used as a carrier gas with a 20 mL/min owrate. The oven
temperature was programmed as follows: 60  C for 2 min,
increasing to 140  C at 5  C/min, and then kept constant at 140  C for
another 6 min. A 50 m 0.32 mm internal diameter fused silica
capillary column coated with 0.2 mm CP-Wax 57 CB was used.
The pH was determined in a slurry 1 part spent solids 5 parts
distilled water at 5  C. Total and volatile solids, Kjeldahl nitrogen,
total phosphorus, cellulose and lignin contents in the feedstock and
digestates were determined as described elsewhere (Poggi-Varaldo
et al., 1997; Muoz-Pez et al., 2008b; Valdez-Vazquez et al., 2006a).
3. Results and discussion
In the rst SSAHF-IV of OFMSW, intermittent venting coupled to
convenient ushing of reactor headspace with N2, allowed for four
cycles of hydrogen generation with no addition of new substrate or
inoculum between cycles (Fig. 1). A fth cycle was attempted, but
no noticeable hydrogen generation was found (data not shown). A
total cumulative production of 16.6 mmol H2/reactor was obtained.
Interestingly, accumulated hydrogen in 4 cycles was 100% higher
than that produced in the rst cycle alone.
Venting and ushing procedures addressed the decrease of H2
partial pressure in headspace which in turn would decrease dissolved H2 concentration in the interstitial liquid of solid substrate.
In such a way, the negative effect of H2 accumulation on further H2
production was minimized or relieved (Mizuno et al., 2000; ValdezVazquez et al., 2005). After each N2 gassing or ushing, the
subsequent production of H2 was lower than the precedent one.
This could be ascribed either to a decrease of biodegradable organic

S357

matter with time or to another form of inhibition determined by

the accumulation fermentation products other than H2.
At the end of incubation, partial pressure of H2 was low and high
concentrations of organic acids and solvents remained in the spent
solids. So, since approximate mass balances indicated that there
was still a moderate amount of biodegradable matter in the spent
solids, we hypothesized that the organic metabolites imposed some
kind of inhibition on further hydrogenogenic fermentation.
As it was mentioned above, cessation of hydrogen production
after the 4th cycle could be due either to the (i) exhaustion of
fermentable organic matter or to an inhibition of the fermentation
possibly caused by the accumulation of (ii) solvents or (iii) organic
acids.
Exhaustion of fermentable organic matter was not likely to be
the cause, since the cellulose-equivalent of biohydrogen harvested
in the rst fermentation was only a fraction of the initial cellulose
(26.2%, Table 1). Furthermore, analysis of washed fermented solids
gave a cellulose content of 18.7% (Table 1). On the other hand,
solventogenic shift could be ruled out since solvent concentration
was lower than that of organic acids (Sum VOA/SumSolvents 11.3,
concentrations in COD-equivalent).
Volatile organic acids may be stimulatory, inhibitory or toxic to
fermentative bacteria, depending upon their concentration levels
(Stewart, 1975; Ezeji et al., 2004; Wang et al., 2008). Inhibition by
VOA is related to the easier transport into bacterial cell of the
undissociated forms of organic acids; once inside the cell the
organic acids dissociate to hydronium ions plus the corresponding
anions, lowering the internal cell pH (Gottschalk, 1986; Jones and
Woods, 1986). Proton uptake uncouples the proton motive force;
this, in turn, causes a rise in maintenance energy in order to keep
the intracellular pH near neutrality. Alternatively, a fraction of
maintenance energy should be used to restore the physiological
balance in the cell, which can reduce the energy used for bacteria
growth and then inhibit the bacteria growth and activity (Jones and
Woods, 1986; Fukuzaki et al., 1990; Zoetemeyer et al., 1982). On the
other hand, if the dissociated species of these soluble metabolites
are present at high concentrations in the fermentation system, then
the ionic strength will increase, which may result in the cell lysis of
hydrogen-producing bacteria (Niel et al., 2003). The uptake of
organic acids may also be linked to a decrease in the available
coenzyme A and phosphate pools; this in turn, would decrease the
ux of glucose through glycolysis (Gottwald and Gottschalk, 1985).
We thus tend to support that organic acids accumulation was
associated to biohydrogen generation cessation in the 5th
fermentation cycle in our work. This is also consistent with ndings
of Wang et al. (2008) who observed that accumulation or spiking
with acetate inhibited H2 production in batch, submerged hydrogenogenic fermentation of glucose. They reported that spiking with
acetate was associated to a lower rate of carbohydrate fermentation
Table 2
Metabolite concentrations in solid substrate anaerobic hydrogenogenic fermentation with intermittent venting and headspace ushing.
Metabolite concentration
(mg/kg wet basis)

Fig. 1. Time course of H2 production in batch mini-reactors: (a) rst batch fermentation of organic fraction of municipal solid wastes; (b) second batch fermentation of
washed, spent solids from the rst fermentation. Vertical arrows indicate ushing of
bioreactor headspace with N2.

Acetone
Ethanol
Acetate
Propionate
Butyrate
Acetone

SSAHF-IV

1st fermentation

2nd fermentation

End of 4th cycle

After washing, before 1st cycle

36
528
2406
1056
4874
36








5
47
26
12
101
5

5  0.6
<DLb
32  4
14  0.2
49  3
5  0.6

a
Notes: batch solid substrate anaerobic hydrogenogenic fermentation with
intermittent venting and ushing of reactor headspace.
b
Detection level.

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K.M. Muoz-Pez et al. / Journal of Environmental Management 95 (2012) S355eS359

Table 3
Hydrogen production in the rst and second solid substrate anaerobic hydrogenogenic fermentations with intermittent venting and headspace ushing.
SSAHF-IVa
1st fermentation
Cycle 1
7.89  1.21
a
b

2nd fermentation
Cycle 2
5.39  0.61

Cycle 3
2.28  0.10

Cycle 4
1.06  0.22

PH cumb
16.62

Cycle 1
0.28  0.07

Cycle 2
2.1  0.22

PH cumb
2.38

Notes: batch solid substrate anaerobic hydrogenogenic fermentation with intermittent venting of reactor headspace.
Cumulative H2 production (mmolH2/mini-reactor).

and a signicant increase of lag time for hydrogen production

onset. In effect, they showed that spiking with ethanol, acetic,
propionic and butyric acids could inhibit the ability of mixed
cultures to generate biohydrogen from de degradation glucose in
submerged batch fermentation tests during the fermentative
hydrogen production. They found that the higher their concentrations, the greater their inhibitory effects. When the added
concentration of organic acids was 200 mmol/L, the inhibitory
effects on the ability of mixed cultures to produce hydrogen were in
this order HAc > HPr > HBu.
Also the increased butyric concentration at the end of fermentation would partially explain the lowering of H2 production in the
last cycles of the rst SSAHF-IV (Table 2), indeed, the ratio A/B was
0.27, where A is the acetic acid concentration and B is the butyric
acid concentration (both in mg COD-equivalent/wet kg). It is known
that the butyric pathway of typical Clostridia yields 2 mol H2/mol
hexose whereas the acetic pathway yields 4 mol H2/mol hexose
(Brock and Madigan, 1991). As a reference, it can be shown that
when both pathways equally contribute to H2 production (50%e
50%), the ratio A/B is 0.79 (on COD-equivalent concentration). So,
the low value of experimental ratio A/B in our work indicates that
the rst batch fermentation was dominated by the lower H2yielding butyric pathway.
Table 2 shows that washing signicantly decreased the
concentrations of organic metabolites in solids; removals of 86.1,
100.0, 98.7, 98.7 and 99.9% of acetone, ethanol, acetate, propionate
and butyrate were achieved.
In the second SSAHF-IV with washed spent solids, two cycles of
H2 production were obtained (Fig. 1, Table 3). In the rst cycle, the
H2 production was relatively low; however, in the 2nd cycle, the H2
generation took off. A 3rd cycle was tried, yet, no hydrogen was
obtained (data not shown). Cumulative H2 production in the 2nd
SSAFH-IV amounted to ca. 2.4 mmol H2/mini-reactor that represented nearly a 14.3% of the hydrogen production of the rst
SSAHF-IV alone.
Even considering that the amount of biodegradable organic
matter in spent washed solids was lower than that in the fresh
OFMSW, an appreciable amount of hydrogen during the second
batch fermentation was observed. This result seems to corroborate
our hypothesis that the presence of organic acids and solvents in
spent solids were related to the cessation of further H2 production
in the 5th cycle of the rst SSAHF-IV. Also, a strategy for maximizing hydrogen yields from OFMSW can be devised, by subjecting
washed spent solids to a second hydrogenogenic fermentation or
recycling them directly to the rst fermentation (mixing OFMSW
with washed spent solids).
The extracts produced in the washing are rich in organic acids
and some solvents and could be post-treated either by photoheterotrophic non sulfur purple bacteria for more hydrogen
generation (Acevedo-Bentez and Poggi-Varaldo, 2008), or in
microbial fuel cells for direct bioelectricity production from the
soluble organic matter (Poggi-Varaldo et al., 2009), or in methanogenic digesters for methane production (Poggi-Varaldo et al.,
2005; Robledo-Narvez et al., 2008). In this way, maximum bioenergy yields could be obtained from the OFMSW.

4. Conclusion
Washing and re-fermentation of spent solids from a rst batch
solid substrate hydrogenogenic fermentation allowed for the
additional generation of biohydrogen, i.e., 14.3% of the biohydrogen
obtained in the rst fermentation. It seems that washing of solids
prevented the inhibition to further H2 production caused by accumulation of organic acids and other metabolites.
Acknowledgments
The authors wish to thank CINVESTAV del IPN and IMCYTDF for
partial support to this research, CONACYT for a graduate scholarship to KMM-P, and COFAA for a fellowship to NR-S. The excellent
help of Mr. Javier Pareja and Mr. Rafael Hernndez-Vera from the
EBRE Group, CINVESTAV del IPN, is gratefully acknowledged. The
authors wish to express their gratitude to the Editor, Guest Editors,
and Referees, for their kind assistance and valuable comments and
suggestions that helped to improve our manuscript.
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Notation
A/B: ratio acetic acid-to-butyric acid, both in COD-equivalent concentrations
BES: bromo-ethanesulphonate
OFMSW: organic fraction of municipal solid wastes
PH cum: cumulative hydrogen production
SSAHF-IV: solid substrate anaerobic hydrogenogenic fermentation with intermittent
venting and headspace ushing
TK: total Kjeldahl nitrogen