i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 8 7 0 4 e8 7 0 7

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Short Communication

Liquid waste from bio-hydrogen production e A

commercially attractive alternative for phosphate
solubilizing bio-fertilizer

Saurabh Jyoti Sarma a, Satinder Kaur Brar a,*, Yann Le Bihan b,

Gerardo Buelna b
a
Institut national de la recherche scientifique, Centre e Eau Terre Environnement, 490, Rue de la Couronne,
Québec (QC) G1K 9A9, Canada
b
Centre de recherche industrielle du Québec (CRIQ), Québec (QC), Canada

article info abstract

Article history: Organic acids produced by commercial phosphate solubilizing bio-fertilizer (PSB) can
Received 18 April 2013 chelate the cations of insoluble phosphates (such as Ca3 (PO4)2) present in soil to solubilize
Received in revised form the phosphate and make it available for plants. Meanwhile, during H2 production (dark
4 May 2013 fermentation) different organic acids are produced as unwanted by-products. Interestingly,
Accepted 6 May 2013 the acid-rich liquid waste of the process has excellent phosphate solubilizing ability (36.12
Available online 2 June 2013 folds increase) and it could be considered as a potential alternative for PSB. Studies have
demonstrated that biodiesel manufacturing waste (crude glycerol) could be used as a
Keywords: substrate for hydrogen production and the secondary waste could be used as a phosphate
Biodiesel solubilizing agent. Thus, this novel approach could: (i) offer a green fertilizer; (ii) improve
Bio-hydrogen liquid waste economic sustainability of bio-hydrogen production and; (iii) could become a financial
Crude glycerol booster for biodiesel manufacturers by offering them a profitable waste management
Organic acid strategy.
Phosphate solubilizing bio-fertilizer Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Waste management reserved.

1. Introduction
Hydrogen is a renewable energy carrier which is postulated to be
one of the clean fuels of future [1]. Water is the only by-product
of its combustion and hence, it has the potential to significantly
reduce the greenhouse gas emission. As a fuel it is suitable for
fuel cells or internal combustion engines and energy content of
H2 is higher than any other gaseous fuels of equivalent weight
[2]. During bio-hydrogen production by dark fermentation,
organic acids such as acetic, butyric and lactic acid are produced
[3], which are generally considered as waste. Actually, produc-
tion of organic acid can drop the pH of the medium to terminate
the process. Similarly, conversion of a significant portion of the
substrate to different organic acids results in reduction of H2
yield. Therefore, organic acid production is considered as the
major bottleneck of dark fermentative H2 production [4]. Inter-
estingly, some of the organic acids can chelate the cations of
insoluble mineral phosphates (present in soil) and release

* Corresponding author. Tel.: þ1 418 654 3116; fax: þ1 418 654 2600.
E-mail address: satinder.brar@ete.inrs.ca (S.K. Brar).
0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2013.05.032
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 8 7 0 4 e8 7 0 7 8705

soluble phosphate to make it available for plants [5]. In fact,

phosphate solubilization by production of organic acid is the
main plant growth promoting mechanism [6] of commercially
used PSB. Thus, organic acid rich bio-hydrogen production
wastes could be a potentially less expensive alternative for PSB.
Moreover, PSBs have relatively shorter shelf-life as they are
actually a preparation of living microorganisms and the organ-
isms must be alive when it is applied to the field. Alternatively,
the performance of the proposed fertilizer does not depend on
the number viable cell it contains and hence, an extended shelf-
life could be expected. Thus, production, packaging, storage and
application of proposed fertilizer should be easier than con-
ventional PSBs.
In general, the liquid waste of dark fermentative H2 pro-
duction using any substrate/any microorganism should be
suitable as the proposed fertilizer. If the proposed strategy
could be integrated into an industrial process, such as bio-
diesel production, apart from hydrogen production, it could
become a profitable strategy for waste management. For
example, as presented in Fig. 1, crude glycerol (CG), the major
by-product (10% by weight) of biodiesel production process [7]
could be used (as a substrate) for bio-hydrogen production and
the final liquid waste could be used as an alternative of Fig. 1 e A schematic representation of the proposed
phosphate solubilizing bio-fertilizers. In fact, crude glycerol is concept.
a hazardous waste and its proper disposal is an additional
expense for biodiesel manufacturers. Therefore, if successful,
the proposed approach may be a huge breakthrough for the flasks were incubated (150 rpm and 28  C) in an orbital
financial/environmental sustainability of biodiesel industry. incubator shaker (Infors HT, Switzerland) and liquid samples
were collected at 3 h interval for the first 12 h (and final
samples were collected after 10 d).
2. Materials and methods
2.3. Analysis
2.1. Hydrogen production
Gaseous samples collected during the investigation were
Dark fermentative hydrogen production experiments were analyzed by a gas chromatograph [8]. Likewise, to determine
carried out using 10 g/L crude glycerol (Rothsay, Canada) as the soluble phosphate concentration, 1 mL of aqueous sample
the only media component. Actually, 10 g/L crude glycerol was collected during the phosphorus solubilization study was
found to be optimum for hydrogen production [8,9]. Likewise, centrifuged (5000 g for 10 min) and the suspended tricalcium
Enterobactor aerogenes NRRL B 407 (ARS, USDA, USA) was used phosphate present in the solution was removed. About 0.1 mL
in this study and all other conditions of this investigation (H2 of the supernatant was collected, diluted 10 times with
production) was similar to our previous report [8]. Briefly, for distilled water and soluble phosphate present in the solution
each experiment, 50 mL CG solution (10 g/L) was taken in was analyzed by using a method described by Murphy et al.
125 mL serum bottle and anaerobic condition was created by [10]. As described by the authors, all glass-wares used in the
passing ultrapure N2 gas through the medium. The bottles analysis were pretreated with concentrated H2SO4 [10].
were properly sealed (using aluminum crimp seal having sil-
icon septum) and autoclaved. Finally, they were inoculated
using 5% (v/v) fresh E. aerogenes culture and incubated 3. Results and discussion
(150 rpm/30  C). Unlike the previous study, in the present case,
H2 gas produced during the process was not periodically Cumulative H2 production using 10 g/L crude glycerol as the
released form the serum bottles used for this purpose [8]. only substrate was found to be 20.61 mmol/L (H2 production
profile is not shown). This value is lower than our previous
2.2. Phosphate solubilization study finding (84.08 mmol/L) using same concentration of the crude
glycerol. It is well known that process mode can strongly in-
To determine phosphate solubilization potential of the spent fluence the hydrogen yield and the observed difference is due
media of bio-hydrogen production process, experiments were to two variable process modes adopted. In the present case, a
conducted using 6 different conical (glass) flasks (125 mL). In closed batch system was used where H2 produced during the
each flask, 50 mg of Ca3(PO4)2 (tricalcium phosphate) was process was accumulated in the headspace of the vessel,
taken and 50 mL of 0%, 10%, 20%, 40%, 60% and 100% (v/v) of whereas, in previous case, produced gas was periodically
the spent media of crude glycerol based bio-hydrogen pro- released from the headspace. Increase in H2 partial pressure in
duction process were added (1 g Ca3(PO4)2 in 1 L liquid). Finally, the headspace is known to negatively affect the production
8706 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 8 7 0 4 e8 7 0 7

and hence the final concentration in the two cases were

Zero waste; high

Pollution/GHG

potential due to
different.

Manufacturing

GHG emission
need separate
emission
Liquid waste generated during crude glycerol bioconver-

production
waste may

additional
treatment

reduction
sion and H2 production process has been evaluated for phos-

biofuel
phate solubilization potential. Fig. 2 summarizes the results of
phosphate solubilization study involving the bio-hydrogen
production waste. In fact, soluble phosphate concentration

sustainable
(other than
Additional
of the spent medium itself was found to be 14.96 mg/L. As a

Production
fertilizer)
being a

of carbon
benefit
control experiment, when only distilled water (0% (v/v) of the

biofuel
waste) was used, soluble phosphate concentration was found

free
No
to be only 1.38 mg/L. In contrast, soluble phosphate concen-
tration as high as 32.06 mg/L had been achieved by using bio-

Includes fermentation
cost (around $0.25/L)c
hydrogen production waste (100%, v/v), which is 23.17 folds

and formulation cost

Production cost

No production cost
(waste by-product)
higher than the control experiment. This finding (23.17 folds
increase within 1 min) is significant in a situation where
conventional PSBs were known to liberate similar amount of
phosphate in a period of 2e3 days [11]. As shown in Table 1,
Zhu et al. (2011) have reported that a newly isolated strain of
phosphate solubilizing bacteria can solubilize 283.16 mg/L of
phosphorus within an incubation period of 11 days [12].

living microorganismb
Only 6 months as it is
However, it should be noted that the authors performed the

not a formulation
phosphate solubilization study using chemically defined

a formulation of
Shelf-life

superior as it is

microorganism
Expected to be
liquid media in optimum laboratory conditions. Hence, the
performance of the strain could be different in real field

of living
application. Moreover, during storage, the cell count of a bio-
fertilizer formulation can sharply decrease [13] and further
reduce its phosphate solubilization ability. Thus, considering
the fact that in the case of biohydrogen production, liquid
Yes, expensive
Chemicals

No, only H2O

chemicals as

components
waste phosphate solubilization is instant as well as it has
required

nothing to do with microbial cell viability, performance of the

required
and CG
present approach is comparable to that of a conventional PSB. media
Table 1 e Possible benefit of proposed technology compared to conventional PSB.

A detailed comparison of the two fertilizers has been pre-

sented in Table 1. Likewise, the phosphate solubilization Bio-hydrogen production
Equivalent to 88,339.22 L

processes was monitored at 3 h interval for 12 h, and phos-

Amount required

waste generated from

25 kg phosphate
(for one hectare

phate solubilization achieved within 1 min was only slightly

to solubilize

of fermented broth

different to that observed after 12 h (data not shown). How-

of land)

ever, after 10 d of incubation enhancement in cumulative

1250 kg of CG

soluble phosphate concentration was recorded to be as high

as 18.59, 25.86 and 36.12 folds for 40, 60 and 100% (v/v) of the
waste, respectively. Interestingly, any significant improve-
ment in soluble phosphate concentration was not observed
period (10e30 days)
solubilization
Soluble phosphorus concentration (mg/L)

Long incubation
Phosphate

60
Within 1 minute of application After 10 d of incubation
solubilization
time

50
Instant

40

30
283.16 mg/L media

49.98 mg/L w aste

solubilization
Phosphate

of incubation)a

20
(after 11 days

b As for average bio-fertilizer.

ability

10
c Sethi et al. (2012) [15].
a Zhu et al. (2011) [12].

0
Control (0%) 10% (v/v) 20% (v/v) 40% (v/v) 60% (v/v) 100% (v/v)
Spent medium of bio-hydrogen production
Bio-hydrogen
Conventional

production

Fig. 2 e Phosphate solubilization achieved using different

Fertilizer

waste

concentrations of bio-hydrogen production liquid waste.

PSB

The control represents soluble phosphate concentration

obtained using only water as the solubilizing agent.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 8 7 0 4 e8 7 0 7
for 10 and 20% (v/v) of the waste. One possible explanation for
such observation could be that a small number of hydrogen
producing bacteria (facultative anaerobe) were still active in
the spent media and they played certain role (e.g. production
of organic acid or enzyme such as phosphatase) in phosphate
solubilization. In the case of 10 and 20% (v/v) of the waste,
such microbial activities were not sufficient (assuming that
only a small fraction of the bacteria was active) for a signifi-
cant response. Moreover, for these two cases, a slight decrease
in soluble phosphate concentration was observed. It might be
due to incorporation of bioavailable phosphate into the
biomass (synthesis of DNA, RNA, phospholipids etc.). How-
ever, these are simple assumptions and further investigation
is required to verify them. Thus, it could be concluded that
bio-hydrogen production waste has excellent potential as a
phosphate solubilizing agent.
In general, a commercial PSB is capable of increasing the
phosphate availability by 25e30 kg per hectare and it is suf-
ficient to increase the product yield up to 20% [14]. Mean-
while, according to the estimated hydrogen production,
liquid waste generated by bioconversion of 1 kg of crude
glycerol would be sufficient to liberate 20 g of phosphate.
Hence, for each hectare, waste generated from 1250 kg of CG
will be required to completely replace a commercial PSB
(Table 1). Therefore, for convenience of application, pre-
concentration of the waste may be required. Although,
phosphate solubilizing ability of bio-hydrogen production
waste has been demonstrated its actual plant growth pro-
moting potential needs to be verified by field scale experi-
ment using food crop.
4. Conclusions
Bio-hydrogen production liquid waste is an excellent phos-
phate solubilizing agent and it has high potential to be a
commercially important alternative of phosphate solubilizing
bio-fertilizer. It can increase the soluble phosphate concen-
tration by 23.17 folds within 1 min and 36.12 folds after 10 d (of
incubation) of its application to a liquid system and this value
may be even better depending on the hydrogen production
process adopted. Unlike traditional phosphate solubilizing
bio-fertilizers, the proposed alternative is not a formulation of
living microorganisms and hence, it is postulated to have
superior shelf-life. Finally and most importantly, the proposed
fertilizer production process could be integrated to biodiesel
manufacturing process (aka biorefinery) to upgrade the bio-
diesel industry to a “high profit-zero waste” industry.
Acknowledgment
SJ Sarma is thankful to FQRNT for providing financial assis-
tance through a doctoral fellowship (MELS). The authors are
8707
also thankful to NSERC, CRIQ and INRS-ETE Canada for
financial support.
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