Journal of Environmental Biotechnology
Vol. 14, No. 1, 65–68, 2014

Technical NoteOptimization of Hydrogen Production byMannitol Utilizing a Bacterium Strain TM1
Atsushi Hongo
Kumagaya Girl’s Upper Secondary School, 2–131 Suehiro, Kumagaya, Saitama 360–0031, Japan
TEL: 03–3918–0511 FAX: 03–3576–0511
E-mail: h-atushi@xk9.so-net.ne.jp(Received; 26 February, 2014/Accepted; 22 July, 2014)
This study investigated hydrogen (H2) production by mannitol fermentation derived from a marine alga. Fermentative H2
production from mannitol (C6H14O6) by a recently isolated bacterium strain TM1 was analyzed under different conditions.
After optimizing cafure condition, maximum yield of 2.9 mol H2/mol C6H14O6 was obtained at 37°C and pH 6.0. DNA
sequences of oxidoreductase and dehydrogenase from strain TM1 showed 98% identity with that of Klebsiella variicola
At-22.
Key words: hydrogen production, fermentation, sea alga, cultivation, mannitol

Introduction
There is a growing interest in issues related to environmental pollution, in particular, the problem of reductionof carbon dioxide levels. Fossil fuels, mainly oil, coal, andnatural gas 1), supply approximately 79.8% of the world’senergy requirement, resulting in the release of massive
amounts of carbon dioxide into the atmosphere. Moreover,
the amount of available fossil fuels is finite, and will eventually be depleted if usage continues at the current rate. Japan
consumes approximately 5.1% of the global reserve of fossil
fuel 2,3), and the vast demand for energy necessitates theirimport, raising additional concerns about energy loss duringtransport.
A concern arising from the ongoing development ofneighboring countries is the increased pollution of oceans.
Industrial waste and algal blooms currently float on the oceansurface, posing serious environmental and health problems.
In addition, along the coastline of Yokohama City on theKanazawa-hakkei sea, which is 1 km long and 60–200 mwide, approximately 1,000 tons (dry weight) of seaweed iswashed ashore each year 4), which is equivalent to about two
million tons for the entire Japanese coastline according toInvestigative report of sea lettuce 2004 5). Incineration of thecollected seaweed has become a valuable organic resourcein Japan: 1 ton of seaweed can be processed to hydrogen(H2) that can be converted into approximately 51.2 kWh ofelectrical energy, that is, approximately 20% of the country’senergy requirement. Thus, exploiting an existing nuisance inthis manner can alleviate the energy crisis as well as reduce
coastline sludge. H2 has recently gained much attention asa sustainable, alternative source of fuel. Unlike the use ofcrops, H2 production from sea algae does not compete withfood production, and can moreover contribute to the removalof nutrient-rich salts that are considered pollutants.
Seaweed is mainly composed of alginic acid and mannitol(C6H14O6) and contains minimal amounts of cellulose. For
example, the constituents of kelp (brown algae) by weight
are alginic acid (7%), C6H14O6 (8%), cellulose (1%), protein(1%), other (4%), and water (79%) 6). Alginic acid is ahighly viscous, minimally degradable material composed ofglucuronic acid coupled to mannuronic acid with a molecularweight of 1,000–100,000. C6H14O6 is a monosaccharide witha molecular weight of 182 and can be used by bacteria asan energy source. H2 production by Enterobacter aerogenes
using C6H14O6 as a substrate has a maximum yield of approximately 1.6 mol H2/mol C6H14O6 7,8), compared to 1 mol H2/mol glucose 9). This difference is largely due to the higher
number of H atoms in C6H14O6 compared to glucose. The
metabolic pathway for H2 fermentation from C6H14O6 10,11) isshown in Fig. 1. 12) C6H14O6 present in seaweed can be readily
extracted by simple grinding 13).
In this study, a bacterial isolate that produce high-yield of
H2 from mannitol as a substrate. Optimization of temperature and pH conditions for maximum H2 production wasperformed. Conditions that promote rapid growth during
seaweed culture were also optimized.

Materials and Methods
Sampling and culture condition of H2-producing bacteriathat uses C6H14O6 as a substrate
Bacteria were collected from the water surface on theMisaki (Miura-shi, Kanagawa, Japan) shore and in theHongo66

Fig. 1. Metabolic pathway of mannitol and possible hydrogen yields. (Hongo, A. 2011, p. 90)
The number of moles of hydrogen produced from 1 mole of a given substance is written under each product.

vicinity of the Iwa (Manazuru-machi, Kanagawa, Japan)
shore; in addition, samples were obtained from sea mudfrom the Yokohama National University marine engineeringside adjustment reservoir and a side channel ditch of thesewage treatment center. Each sample was inoculated using asterile platinum loop and cultured in a test tube in semisolid
nutrient medium (10 ml) containing 10 g/L C6H14O6, 10 g/L
casamino acid, 10 g/L dried yeast extract D-3, 2 g/L agar,(pH 6.0) at 37°C for 24 h. The test tube served as a simple
gas generator that allowed quantification of the gas generated (Fig. 2). A small volume of the culture was transferred tomedium containing 10 g/L C6H14O6, 20 g/L casamino acid,20 g/L trisodium citrate dihydrate, 5 g/L NaCl, 15 g/L agar,
and 10 g/L dried yeast extract D-3, 2 g/L agar, (pH 6.0)
and the cultures were incubated at 37°C for 24 h. H2 sulfide
produced as an off-gas was measured using a gas detector
tube (No. 4LT; Gastec Corp, Ayase-City, Japan). Bacterial
isolates that produced high levels of H2 sulfide were excluded.
Optimization of culture condition
Experiments were performed to determine the optimal
temperature and pH for bacterial culture. Isolated bacteria
were cultured in approximately 10 ml of semisolid medium
for 24 h in a simple vapor generator. The pH of cultures wasadjusted to 5.0, 5.5, 6.0, 6.5, or 7.0 with phosphoric acid andsodium hydroxide. Cultures at each pH were incubated at35°C, 37°C, 40°C, 42°C, and 45°C.

Fig. 2. Hydrogen production apparatus.
(a) Fermentative gas collection tube; and (b) test tube containing substrate and cell culture.

Optimization of Hydrogen Production by Mannitol Utilizing a Bacterium Strain TM167

sodium hydroxide was used to remove carbon dioxide fromthe generated gas.
Oxidoreductase and dehydrogenase DNA sequences
Bacteria screened in Section 2.1 were identified by sequencing
the PCR products for the 16S rRNA gene. Sequencing wasperformed by Bio Matrix Research Inc. (Chiba, Japan).
Portions of the DNA sequence were registered in DDBJ
(accession number AB746174). The sequence data were usedto perform a homology search of the DNA Data Bank ofJapan (DDBJ; http://www.ddbj.nig.ac.jp/) using the Basic
Local Alignment Search Tool to identify the isolates.
Fig. 3. Batch culture apparatus. (Hongo, A. 2011, p. 91)(a) pH-measuring device (Fine pH controller SERIES-BFD-02,NISSIN TYPE CE108C-1.8BG); (b) temperature-measuring
device (SHINO.LT100); (c) culture vessel; (d) stirring device(ASONE MAGNETIC STIRRER HS-4SP); (e) NaOH vessel;(f) electric scale (Sartorius BJ6100); and (g) measurement
container.

Batch cultivation
Bacterial isolates were cultivated in 500 ml liquid medium
consisting of 10 g C6H14O6, 10 g/L casamino acid, 10 g/L dried
yeast extract D-3, 0.3 g/L CH2CH(NH2)COOHHClH2O, and
0.3 g/L fumaric acid. Batch cultivation was carried out at pH
6.0 and 37°C, which were determined as the optimal conditions for bacterial growth. H2 production was quantified bydetermining the change in the absorbance of the culture at560 nm, and by Hitachi RI Monitor detector and 655A-30
analyser (Hitachi High-Tech, Tokyo, Japan), as well as bygas chromatography (Shimadzu GC-3BT; Shimadzu Corp.,
Kyoto, Japan). Analysis of organic acids, using by liquid
chromatography using a GL-C610H-S column (columntemperature, 40°C; carrier, 0.1% phosphoric acid; flow rate,0.5 ml/min). The batch cultivation apparatus is illustrated inFig. 3. 14) For the downward displacement of water (Fig. 3e),(a)Results and Discussion
Isolation of H2-producing bacteria
The TM1 and TMS8 strains were isolated after repeatedtransfer of medium. Results from experiments determining
optimal temperature and pH conditions for TM1 and TMS8
were shown in Fig. 4(a) and 4(b), respectively. H2 production
was highest at 37°C and pH 6.0 for both TM1 and TMS8. A
decline in H2 production was observed at temperatures above
37°C. Maximum H2 production was nearly twofold higher in
TM1 than in TMS8.
Batch cultivation
Average of batch cultivation of TM1 hydrogen production resulted is 2900 mL/L, and maximum, minimum,and mean yields of 2.9, 1.8, and 2.1 mol H2/mol C6H14O6,respectively (Fig. 5). The maximum and mean absorbancevalues at 560 nm were 1.2 and 1.1, respectively. The lattercorresponded to the lowest H2 yield, indicating that there
was likely to be correlation between bacterial concentrationand gas production. The bacterial concentrations calculated
from the absorbance measurements are shown in Fig. 5.
Liquid chromatography showed that TMS8 and TM1 mainly
produced lactic and butyric acids, respectively. However, in
the results for TM1, an overlapping C6H14O6 peak prevented(b)Fig. 4.

Experimental results of the optimisation of culture conditions of TM1 (a) and TMS8 (b).

Hongo68

with high hydrogen yield most in this laboratory. The isolatedbacterial strain TM1 has been found that high hydrogen yieldin the most bacteria was discovered until now.

ReferencesFig. 5. Hydrogen production TM1 bacteria and Change in absorbance of TM1 bacteria over time.

the precise molecular quantification of butyric acid.
Analysis of TM1 oxidoreductase and dehydrogenase DNAsequences
DNA sequences of TM1 oxidoreductase and dehydrogenase was performed showed 98% identity with Klebsiellavariicola At-22 enzymes.

SummaryIn this study, a new strain of bacterium, TM1, was isolated
and was found to produce H2 using mannitol as a substrate.
The optimal conditions for obtaining a maximum H2 yieldof 2.9 mol H2/mol C6H14O6 with the bacterial culture were atemperature of 37°C and pH of 6.0.
When using C. butyricum, hydrogen yield is 2.3 mol H2/mol According to “The Bacteria” in Wood, WA in the fermentation hydrogen production using the substrate glucose, asubstrate mannitol According to the article by S. Tanisho, inthe fermentation hydrogen production using hydrogen yield
was 1.4 mol H2/mol 13). When used glucose as a substrate,maximum hydrogen yield 2.4 mol H2/mol HN001 is a fungus1) Energy white paper. 2008. Japan Ministry of Economy, Tradeand Industry.
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