Responses to the Reviewers' comments of the submission: Metabolic flux analysis of

an anaerobic mixed culture with H2 production (BBEN-D-13-00567)

To the editor: We would like to thank the reviewers for the constructive comments and for
taking the time to read our manuscript. We have addressed all of the reviewers concerns
and made suggested changes to improve the manuscript. Reviewers comments are first
listed, followed by authors responses in blue. Changes made in the manuscript are
presented in red.

Reviewer #1:
1. Biological hydrogen production is cleaner and consumes less energy than more widely
used chemical hydrogen production processes. However, numerous studies have reported
low hydrogen production yields by anaerobic microorganisms grown on glucose. To better
understand and analyze cellular processes involved in hydrogen production, R. A.
Gonzalez-Garcia and E.I. Garcia-Pena have demonstrated a simplified metabolic model that
can describe H2 synthesis by a mixed culture of anaerobes. The model presented in this
study is useful for developing metabolic engineering strategies that may contribute to
improvements in hydrogen productivity and thus lead to a more efficient and economical
process.
We appreciate that the reviewer recognizes that the model could be useful for developing
metabolic engineering strategies as well for its potential applications to improve H2
productivity.
2. Computational tools to analyze cellular metabolism have been developed for more than
two decades. The authors use two such tools in this study, namely metabolic flux analysis
(MFA) and flux balance analysis (FBA), to calculate the intracellular flux distribution by a
mixed culture of hydrogen-producing anaerobes in various theoretical and experimental
scenarios. These two computational techniques (MFA and FBA) have been previously
described many times in the literature for analyzing cellular metabolism; still, this reviewer
found the detailed explanation of these techniques in the materials and methods section to
be fresh, clear, and succinct. Clearly, the authors possess a solid understanding of flux

analysis in the context of systems biology. Furthermore, the results demonstrate the utility
of the metabolic model developed in this study for metabolic flux analysis and other
stoichiometric modeling techniques. In a simulated acetic acid or butyric acid fermentation
case, the relationship between calculated values for hydrogen production as compared to
the theoretical maximum was found to be 85% and 78%, respectively. For an experimental
mixed fermentation, the calculated H2 production rate (176 mmol/h) was found to be 79%
of the experimental value (223 mmol/h). This reviewer mostly agrees that the MFA results
can be considered adequate for describing carbon flux distribution of the mixed culture
during hydrogen synthesis. The metabolic model presented here will certainly be a good
reference and starting point for related studies in the future. However, it is suggested that
the authors include some discussion about limitations of the model and possible
modifications that may lead to more accurate results.
Discussion about the limitations of the model and possible modifications has been included
in the revised version of the manuscript in the Results and discussion section (Page 11, line
24; page 20, line 12).
3. The authors gave some interpretation of the MFA results for explaining differences
between the acetic acid, butyric acid, and mixed fermentation cases. This reviewer agrees
that the discussion of these cases provides useful information about the metabolic processes
involved in hydrogen production. Several insightful and non-obvious metabolic engineering
strategies are proposed for improving hydrogen production in a mixed anaerobic cultures: i)
enhancing activity in the pentose phosphate pathway, ii) eliminating by-products which
consume NADH such as ethanol and/or lactate, and iii) redirecting metabolic fluxes to a
mixed fermentation where acetate and butyrate are produced.
We thank the reviewer for the observation.
4. The manuscript is fairly well written in most places, but this reviewer did locate some
grammatical errors and awkwardly worded sentences that will distract the reader. The title
should be edited and a suggestion is "Metabolic flux analysis of an anaerobic mixed culture
with H2 production."
The title was modified according to the reviewer suggestion.

5. A careful revision of the whole manuscript is suggested. The following provides a sample
of some suggested changes for the abstract only.
English language was revised through the entire manuscript, but mainly in the abstract
section.
1. L2-3: The second "sentence" is actually a sentence fragment and should be combined
with the first sentence.
We have made the appropriate change.
2. L5-6: For better clarity, change to "the calculated H2 production rate (176 mmol/h)
was shown to be 79% of the experimental H2 production rate (223 mmol/h)."
We have made the appropriate change.
3. L7-8: "Acetic, butyric, and mixed fermentations" is brought in abruptly and it may help
to explain in a very brief way what these terms mean as they may be unfamiliar to some
readers.
We clarified this aspect in the abstract of the revised version of the manuscript.
4. L8: NADH reduction sounds like NADH is being reduced which does not make sense.
This could be changed to "NADH generation" or something similar.
We have made the appropriate change (Changes are highlighted in red in the manuscript).
5. L13: I think this should read "FBA showed that at low acetate or butyrate production
rates"
We have made the appropriate change.
6. L17: Change to "anaerobic cultures"
We have made the appropriate change.

Reviewer #2: Summary

The authors have constructed a simplified flux-based model of H2 production in anaerobic
mixed cultures. Model simulation agreed with an experimental observation. The major
problem seen with this approach is that the model does not accommodate for different
mixed culture compositions and cannot be used for metabolic engineering purposes because
the mixed culture is largely undefined. Further, the model was not used to design
experiments to improve H2 production.
We thank reviewer comment. However, we would like to disagree with the reviewer in this
regard. As we have stated in the manuscript, the aim of our work was the development an
accurate model both to predict H2 production yields and to describe the internal flux
distribution in a mixed anaerobe culture so that the metabolic processes involved in H2
production could be understood. Thus, the knowledge generated can be used for further
experiments.
Regarding the reviewers concern about the validity of the model in relation with the
populations composition, we have described in the Materials and Methods section
Metabolic model, the concept of universal bacteria. This concept has been previously
discussed by Changati et. al. 2011. They used a similar strategy for describing flux
distributions in anaerobe communities. Certainly, populations in mixed cultures changes
through time. However, at the time when all calculation and experimental data were taken
(maximum H2 production rate), it is expected for the H2 producers to predominate over the
other microorganism. Additionally, we have described the population composition in our
culture at that stage. Moreover, the report of Stolyar et. al. 2007 stated that during
degradation of organic substrates, communities present in a microbial culture perform
syntrophy. This process has been widely report for H2 and methane producers. Thus, the
use of a single model for describing flux distribution in mixed population is valid, by
assuming the pseudo-steady state (which is the basis of FBA and MFA) and when it is
known that one of the population dominates on the culture, as shown in our work. Also, we
consider that the use of mixed culture is important for many applications. The main
contribution of our experimentally validated model is that it will provide the opportunity to
systematically optimize microbial communities for practical applications through the

engineering of the individual species, addition of new species to existing communities and
manipulation of the community composition and environment. Additionally, we evaluated
the robustness of the model with other reported data and a new Table (Table 5) was
included in the revised version of the manuscript. There exist in the literature some reports
where genome-scale models have been used to identify potential strategies for engineering
metabolism in communities. Typically, engineering communities can involve: 1)
engineering the metabolism of individual microbes in defined microbial communities; 2)
manipulating the community by adding new microbes (e.g., bioaugmentation); and 3)
engineering the environment by introducing substrates that allow a specific species of
interest to dominate (e.g., bioremediation). (Mahadevan and Henson, 2012).

[Mahadevan R. and Henson M. A. 2012. Genome-based modeling and design of metabolic

interactions in microbial communities. Computational and Structural Biotechnology
Journal 3, http://dx.doi.org/10.5936/csbj.201210008.]
Finally, the reviewer points out that metabolic engineering might me limited due to the lack
of information about the culture. However, we would like to disagree with the reviewer in
the aspect that the mixed culture is largely undefined. Our most recently results showed that
the bacterial population during the batch mode operation was dominated by phylotypes in
the Firmicutes (93.1%) and Actinobacteria phyla (5.9%). After 24th hour of the batch
culture, the mixed microbial consortium in H2 production systems was composed mainly
by the Prevotellaceae family (66.7%) (Esquivel-Elizondo, 2012). Phylotypes within this
family are constituted by acetate and H2 producers (Shah and Collins, 1990). Quantitative
PCR (qPCR) assays showed that heat pre-treatment inhibited the proliferation of
methanogens and homoacetogenic bacteria, two important H2 scavenging groups. The
characterization of dominant community members allows obtaining a representative model
that adequately captures the collective metabolism of the mixed culture (Mahadevan and
Henson, 2012). Also it have been demonstrated that theres functional redundancies in key
metabolic pathways that synthesize same byproducts which contribute to the robustness of
community function.

Major Points
1. The Abstract will require additional editing for proper English and sentence/paragraph
structure. The past verb tense should be used consistently when describing observations.
Currently, there are too many errors to list individually. These problems were not present in
the rest of the manuscript.
We appreciate the observation of both reviewers regarding this point. We have revised both
the abstract and the entire manuscript and corrected them when required.
2. In the abstract, it is also not clear what experimental conditions are being used. In
addition, can the model give successful predictions under multiple experimental
conditions? Hydrogen production rates are being reported as mmol/h. Often, it is
advantageous to report production flux as mmol/(h*gDCW).
We agree with the reviewer in this point. Thus, we have changed the units of flux rates in
the entire document properly as requested by the reviewer. Additionally, we have corrected
the abstract to include the experimental condition of the performed fermentations.
Regarding the reviewer concern about, we have discussed in the manuscript the validity of
the model to be used in several conditions when mixed cultures produce H2. At this point, it
should be noticed that he model is only valid for those mixed cultures in which H2
production occurs under the metabolic pathways included. Additionally, we have included
one table in which we used reported data for similar fermentations to feed the model and
we calculated and compared with the predicted yield.
3. From where was the mixed culture obtained? Why was this culture worth investigating?
The inoculum was obtained from an anaerobic digester periodically fed with ground fruit
and vegetables residues. The inoculum was heat pre-treated at 80 C for 35 minutes in order
to inhibit methanogens and non-spore forming bacteria, as previously reported (GarciaPea et al., 2009).
Our previous works showed that with this mixed culture it is possible to obtain high
hydrogen molar yields. Results showed that the heat pretreated inoculums (35 C) reached

the highest molar yield of 2.85 mol H2/mol glucose (0.014 L/h), which corresponds to the
acetic acid pathway (Garcia-Pea et al., 2009).
4. It is not clear what happens if another organism in the mixed culture begins to dominate.
Do the model predictions hold true no matter the composition of the mixed culture? This is
seen as the major weakness of the manuscript and needs substantial explanation.
We understand the reviewers concern about it and we have given a response for that in the
response to his first comment. Additionally, we have expanded the Material and Methods
for a better understanding and we have included an additionally paragraph in the Results
and Discussion section.
The discussion was included in the revised version of the manuscript as follows:
Previously reported data for H2 production was taken from literature to feed the model.
The results, regarding H2 production rates are shown in Table 5. As seen, the model
accurately predicts similar values to those reported previously. Differences among data
might be due to the use of the same biomass molecular formula in all calculation since such
information was not found in the cited works. Also, the model considers only the pathways
involved in H2 production since the mixed culture used in this work was heat-treated
according to Materials and Methods to eliminate methanogens. However, since average
similarity was about 83 % it can be considered that the model possesses enough robustness
to be used for calculating yields and thus the flux distribution in anaerobic H2 producer
systems.
5. Figure 1. Reaction 12 is not clear because it does not seem to involve a Fe_red substrate.
In addition, Table 1 does not include Fe_ox as a metabolite. It is not understood why this
must be the case.
We disagree with the reviewer at this point. We consider that Figure 1 is clear since clearly
show the process of Ferredoxin oxidation from Fered to Feox by the coupled reduction of
NADH.
Regarding the reviewers observation for the absence of Feox in Table 1, we have explained
in the Materials and Methods section that when coupled pairs appear in a reaction, in this

case Fered and Feox, it was necessary to eliminate one of them (Feox) to avid redundancies in
the linear equations system.
6. p.16. The authors talk about possible metabolic engineering strategies to improve H2
production, but how is this possible when the culture is a largely undefined mixed culture?
We thank reviewer comment. We have discussed it above in the response to the first
comment of the reviewer.
The microbial dynamics and identification of the microorganisms related to the highest H2
yield in a process in a batch and continuous culture under different conditions were recently
study. The characterizations of dominant community members allow obtaining a
representative model that adequately captures the collective metabolism of the mixed
culture (Mahadevan and Henson, 2012). Also it have been demonstrated that theres
functional redundancies in key metabolic pathways that synthesize same byproducts which
contribute to the robustness of community function.
The use of mixed culture is important for many applications. The natural environments
based its success different strategy based on synergistic combinations of different microbial
species that collectively achieve the desired task. The development of new technologies
needs to exploit the natural metabolic capabilities of different species and made bioprocess
highly adaptive to changes in the environmental conditions.
Overall, it seems that only a single data point was validated (with 79% accuracy). The
model should also be validated using mixed cultures of different compositions. A variable
for culture composition may need to be built into the model to have truly accurate model.
This would indeed be novel.
We have discussed previously the validity of the model and we have provided references to
support it. Additionally, we have included a Table in which the accuracy of the model is
validated by feeding the model with data reported for other authors.
Minor Points
p.5, line 7. "FMA" should be "MFA"
We appreciate the reviewer observation regarding this point and we have made the
requested changes.

p.5, line 22. What is "pyro-sequencing"? Are there any references to these results? Where
can they be found?
The Pyrosequencing analysis was the first second-generation DNA sequencing platform to
be commercially available; it was developed by Roche 454 Life Science (Margulieset al.,
2005). The main advantage of 454-pyrosequencing is its read-length, which is typically 400
bp (Shendure and Ji, 2008). Pyrosequecing additionally enable the detection of many
microbial species and the entire information about the microbial community structures can
hardly be revealed. Pyrosequencing analysis is more accurately and faster than the clone
library method which is time-consuming and expensive and the number of sequences that
can be generated by is very limited (DeSantis et al., 2007).
The second-generation high-throughput sequencing new technologies have been developed
to better elucidate the characters of microbial community more completely and accurately
(Roesch et al., 2007). Additionally to overcome some limitations of the techniques like
PCR-DGGE method were one band may contain more than one species (Muyzer and
Smalla, 1998). Thus, to more fully investigate the microbial community, more sensitive
detection is needed.
This technology was first applied by other groups to study the composition of microbial
communities found in the ocean (Sogin et al., 2006), soil and recently in the microbial
community structures in different wastewater treatment plants (Hu et al., 2013).
Hu M., Wang X., Wen X., Xia Y. 2012. Microbial community structures in different
wastewater treatment plants as revealed by 454-pyrosequencing analysis. Bioresource
Technology 117, 7279.
In our previous published work we reported that the mixed microbial inoculum generated
from the anaerobic digester were distributed among three major phyla, identified by
pyrosequencing: Firmicutes (89.5%), Actinobacteria (6.9%), Bacteroidetes (2.3%), along
with other phyla at minor predominance. Firmicutes are well-known to be fermenters and
syntrophic bacteria that can degrade volatile fatty acids, such as butyrate and its analogs.

Within the phylum Firmicutes, Bacilli (76.1%) and Clostridia (13.3%) form the major
classes (Garcia-Pea et al., 2011).

100%

80%

60%

Relative abundance at phylum level

40%

Tenericutes
Proteobacteria
Actinobacteria
Other
Bacteroidetes
Firmicutes

20%

0%

Our most
recently results showed that the bacterial population during the batch mode operation
(B0.47) was dominated by phylotypes in the Firmicutes (93.1%) and Actinobacteria phyla
(5.9%) as shown in the Figure. After 24th hour of the batch culture, the mixed microbial
consortium in H2 production systems was composed mainly by the Prevotellaceae family
(66.7%) (Esquivel-Elizondo 2012). Phylotypes within this family are constituted by acetate
and H2 producers (Shah and Collins, 1990). In this work the microbial dynamics and
identification of the microorganisms related to the highest H2 yield in a batch and
continuous culture was performed. The study was developed under batch (usual pH2 of 0.47
atm) and continuous culture conditions (low pH2 of 0.043 atm). Some of the results
obtained with the pyrosequencing analyses are summarized in the Figure.

Figure. Taxonomic levels identified during the complete fermentation (i.e. batch and
continuous culture) by the 454-Pyrosequencing analysis.

According to the literature, 5 genera identified in our fermentation process produce acetic
acid with H2 as a major end products. These HPB are among the most abundant phylotes at
the genus level identified in this study.
p.6, line 24. It should be "Mavrovouniotis". In addition, it should be specified what this
algorithm does.
We appreciate the reviewer observation regarding this point and we have made the
requested changes. Additionally, we have expanded the Material and Methods section to
provide a brief explanation of the algorithm.
Fig. 6. The axis titles need to be corrected.
We appreciate the reviewer observation regarding this point but we do not understand the
reason why the reviewer asks to change the axis. This contour graph represents hypothetical
mixed fermentations and shows the effect of Acetate and Butyrate yields when the culture
is grown on glucose on the expected H2 yield. The units therefore should be as indicated, in
mol/molGlucose. However, we have changes the units for flux rates in the entire manuscript to
mmol/(h*gDW) as previously requested by the reviewer.