Bioresource Technology 154 (2014) 254–259
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Photo-biological hydrogen production by an acid tolerant mutant of Rhodovulum sulfidophilum P5 generated by transposon mutagenesis Jinling Cai a,⇑, Guangce Wang a,b,⇑ a b
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, PR China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong, PR China
h i g h l i g h t s Transposon mutagenesis was used to enhance H2 yield of a photosynthetic bacterium. A transposon mutagenesis library of Rhodovulum sulfidophilum P5 was constructed. Mutant TH-102 had higher aciduric and temperature resistant ability. TH-102 can produce H2 at dark fermentation effluent environment (pH 5.5 and 35 °C). In continuous culture, H2 yield and rate were 17 and 15-fold higher than the WT.
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 10 December 2013 Accepted 12 December 2013 Available online 22 December 2013 Keywords: Hydrogen production Photosynthetic bacteria Acid tolerant Transposon Mutation
a b s t r a c t Most of the photosynthetic bacterial strains exhibit optimum hydrogen production at neutral initial pH, and lower initial pH resulted in a sharp decrease in hydrogen yield. Thus, screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. To obtain acid tolerant mutants, a Tn7based transposon was randomly inserted into the genomic DNA of Rhodovulum sulfidophilum P5. An acid tolerant mutant strain TH-102 exhibited increased hydrogen production in acidic environment (pH 4.5– 6.5) and at higher temperatures (35 and 37 °C) than the wild-type strain. At pH 5.5 and 35 °C, the mutant strain TH-102 continuously produced hydrogen. The hydrogen yield and average rate were 2.16 ± 0.10 mol/mol acetate and 10.06 ± 0.47 mL/L h, which was about 17.32 and 15.37-fold higher than that of the wild-type strain, respectively. This acid- and temperature-tolerant mutant strain TH-102 could be used in a cost-effective hydrogen production process employing both dark fermentative and photosynthetic bacteria. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Due to the limited availability of traditional energy from a nonrenewable reservoir and as a result of ever-growing energy demand, there is an increasing interest in the search for renewable energy sources to meet the current and future energy requirements. Numerous alternative and renewable energy resources have been explored. Among them, hydrogen is an attractive potential alternative energy source due to its non-polluting and environmental-friendly nature, and currently, sustainable biological hydrogen production is under active investigation. The low yield and production rate are still major barriers for commercial biological production of hydrogen. It has been
⇑ Corresponding authors at: Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, PR China. Tel./fax: +86 022 60601305 (G. Wang). E-mail addresses: [email protected] (J. Cai), [email protected] (G. Wang). 0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.057
estimated that a hydrogen yield of 8 mol H2/mol glucose would be sufficient for practical production (Keskin et al., 2011). However, the hydrogen yield of all the principal biological hydrogen production methods such as direct and indirect biophotolysis, photo fermentation, and dark fermentation, is relatively below this benchmark. In some studies, integration of dark and photo fermentations has been reported to produce yields of up to 7.1 mol H2/mol hexose (Asada et al., 2006; Chen et al., 2008) (even up to 8.3 mol H2/mol hexose (Kim et al., 2006)). Thus, further investigations are required for the development of these systems for practical purposes. The combined use of dark-fermentative bacteria and photosynthetic bacteria, which is a promising hydrogen production approach, can markedly enhance hydrogen yield. Most of the dark-fermentative bacteria can produce hydrogen at high rate, but cannot degrade organic compounds completely due to thermodynamic limitations. The maximum theoretical hydrogen yield of dark fermentation is relatively low, ranging from 2 to 4 mol/mol hexose according to the composition of the organic acids produced.
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
As a result, the dark-fermentative hydrogen-producing effluent primarily contains organic acids (mainly acetate and butyrate) and alcohols (Lee et al., 2010), mostly in their undissociated forms, which can cross the cell membrane at a low pH and can adversely affect hydrogen production (Wang et al., 2008). Furthermore, the presence of organic acids and alcohols in the residual medium poses a disposal issue. However, the organic acids could be further metabolized to produce additional hydrogen by photosynthetic bacteria under photoheterotrophic conditions, which increases the theoretical hydrogen yield from 4 to 12 mol H2/mol glucose and reduces the organic content of the final residual waste. Several studies have reported that the combined use of both dark-fermentative and photosynthetic bacteria could achieve higher hydrogen yields from various substrates, when compared with the hydrogen yields obtained using either of these bacteria (Lee et al., 2010; Liu et al., 2009; Patel et al., 2012). Thus, the combined use of darkfermentative and photosynthetic bacteria may overcome individual limitations and exhibit greater advantages. Low pH of the dark-fermentative effluent inhibits hydrogen production and even growth of the photosynthetic bacteria, which is one of the major barriers for the stable operation of the integrated process. The pH for dark fermentative effluent and optimum pH for hydrogen production are in the acidic range of 4.5–5.5 (Karadag and Pahuakka, 2010; Luo et al., 2010; Masset et al., 2012; Sigurbjornsdottir and Orlygsson, 2012). However, the optimum pH for hydrogen production by most of the photosynthetic bacteria is neutral (Nath and Das, 2009; Wu et al., 2012; Yang et al., 2012; Zhu et al., 2010) to weak alkaline (Cai et al., 2012; Cai and Wang, 2012; Pandey et al., 2012). It has been observed to be necessary to control the pH of the fermentation effluent for further production of hydrogen by photosynthetic bacteria (Ljunggren et al., 2011). However, controlling the pH of the dark fermentation process requires large amounts of alkaline solution, which not only presents an economic burden, but also increases the concentration of salts in the treated effluent, requiring adoption of an efficient way to reduce the amount of alkaline solution. Hence, it is necessary to achieve acid tolerant remediation of fermentation effluent, which could reduce alkali consumption, as well as increase the hydrogen yield. Screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. These bacteria are not only acid tolerant, adapting to acid stress and alleviating organic acid inhibition, but are also beneficial for hydrogen production as well as for the integration of dark and photo fermentations. Although the traditional isolation strategy for screening acid-tolerant photosynthetic bacteria for hydrogen production is effective, it is time-consuming. Some powerful genetic engineering approaches could be employed to improve the hydrogen yield (Hallenbeck et al., 2012; Kim et al., 2006). Among them, transposon technology, carrying antibioticresistance genes, is an excellent indispensable tool in bacterial genetics, especially for those bacteria whose genetic system has not yet been developed (Ma et al., 2012; Cai and Wang, 2013). Transposon mutagenesis is an important genetic tool for the creation and characterization of insertion mutants, and has been used to carry out insertional mutagenesis screening of mutants of photosynthetic bacteria which could produce high hydrogen yield, such as Rhodovulum gelatinosus (Vanzin et al., 2010), Rhodovulum capsulatus (Ma et al., 2012), and R. sulfidophilum P5 (Cai and Wang, 2013). Furthermore, the use of transposon mutagenesis technology to create a mutant bank of R. sulfidophilum P5 could significantly contribute to enhancing its hydrogen production as well as our understanding of the mechanism of hydrogen production. To date, biological hydrogen production by photosynthetic bacteria in fresh conditions has been well developed. Hydrogen production by marine photosynthetic bacteria from marine wastewater has been attracting increasing attention. Some studies
255
have reported hydrogen production by marine photosynthetic bacteria, such as Rhodobium marinum (Anam et al., 2012), R. sulfidophilum P5 (Cai and Wang, 2012), and Rhodovulum spp. (Matsunaga et al., 2000), as well as marine mixed phototrophic bacterial consort (Cai et al., 2012). However, most of the investigations have been mainly focused on optimizing the basic parameters, including the operating conditions, substrate selection, immobilization of photosynthetic bacteria cells for a higher retention time, etc. Only a few studies have attempted to improve the hydrogen yield of photosynthetic bacteria through molecular biology methods (Ma et al., 2012). Thus, the main purpose of the present study was to screen acid-tolerant photosynthetic bacteria producing high hydrogen yield, which could be used in the integrated dark–photo fermentation process for hydrogen production. 2. Methods 2.1. Bacterial strain and culture medium The transposon library of R. sulfidophilum P5 was created according the forward research (Cai and Wang, 2013). Transposition was performed using the GPS mutagenesis system (New England Biolabs Catalog NO E7101S), according to the manufacturer’s instructions. The transprimer donor used was pGPS3, which carries kanamycin resistance. The acquired mutants were stored at 80 °C in 15% glycerol. R. sulfidophilum strains were grown on RCVBH medium (Cai and Wang, 2012). The RCVBH medium contained the following: 20 mmol/L acetate, 10 mmol/L glutamate, 20 g/L NaCl, 75 mg/L CaCl2 2H2O, 120 mg/L MgSO4 7H2O, 10 mmol/L KPO4 buffer, 1 mg/L thiamine hydrochloride, 20 mg/L sodium ethylenediaminetetraacetic acid, and 1 mL/L trace element solution (per 100 mL of deionized water (dH2O): 280 mg H3BO3, 75.2 mg NaMoO4 2H2O, 159.2 mg MnSO4 H2O, 3H2O, 4 mg Cu(NO3)2, and 24 mg ZnSO4 7H2O). For basic growth, the pH was adjusted to 8.0. 2.2. Screen and characterization of acid tolerant mutant To verify the hydrogen production ability of the mutants, hydrogen production profiles of different mutant strains were grown under acidic condition (pH 4.5) using 50 mL anaerobic tube (20 mL working volume). Among all the mutants, only 9 mutants could produce hydrogen under acidic condition (pH 4.5). These nine acid tolerant mutant strains could evolve hydrogen were further analyzed the nature of hydrogen production. A total of 50 mL of pre-cultured cells (OD660 = 0.8–1.0) were harvested by centrifugation and inoculated into 500 mL of RCVBH medium. Argon gas was purged into the bioreactors to create anaerobic conditions. The bioreactors were stirred at 150 rpm at 30 °C. The light intensity was 100 lmol photons/m2 s. For further analysis of the hydrogen production nature by the acid tolerant mutant strain TH-102, two series of test batches (of 500-mL working volume) were cultured under anaerobic conditions in RCVBH medium, with the WT strain used as the control. For the initial pH tests, the pH was varied from 4.0 to 9.0. For the culture temperature test, the temperatures ranged from 30 to 40 °C. For semi-batch hydrogen production, the mutant strain TH-102 and the WT strain in exponential growth phase were harvested and inoculated into 500-mL culture medium. Initially, the reactors were operated in batch mode for 5 days to accumulate biomass for continuous operation. Thereafter, 125 mL of the exhausted medium was withdrawn and replaced with fresh medium. The reactor was operated under the same conditions as described
256
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
earlier. The culture temperature was 35 °C and the initial pH was 5.5, reflecting the environment of dark-fermentative hydrogenproducing effluent. 2.3. Stability of acid tolerant mutants The stability of the mutant strain TH-102 was by preparing five subcultures in non-selective media, and then inoculating the cells into kanamycin media (50 lL/mL kanamycin sulfate). The transposon fragment was confirmed by PCR using primers derived from transposon sequence (P1: 50 -TTAAGGATTATTTAGGGAAG-30 ; P2: 50 -ACAATAAAGTCTTAAACTAG-30 ). The PCR program was as follows: 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. After completion of 30 cycles, a 10-min extension was run at 72 °C. The identity of the generated transposon fragment was further verified mutation stability of the mutant strain TH-102. 2.4. Analytical methods When hydrogen production ceased for each tests, hydrogen and cell dry weight were analyzed. Hydrogen was analyzed by gas chromatography (GC-2010, Tengzhou, China), equipped with a 2-m stainless steel column packed with Porapak DMCS (60/80 mesh) and a thermal conductivity detector. The operating procedure was according to that reported in a previous study (Cai and Wang, 2012). The cell dry weight was determined as described previously (Cai et al., 2012). 2.5. Statistical analysis All the results were presented as mean ± standard deviation (SD) values of the triplicates for each experiment. Statistical analyses were performed using the IBM SPSS Statistics 19 package (IBM Co., Armonk, New York, USA). T-tests (a = 0.05) were used to determined whether significant differences were exist between various treatment groups. 3. Results and discussion 3.1. Acid tolerant mutants screen About 350 mutant colonies were acquired from forward transposon mutagenesis system (Cai and Wang, 2013). To screen acid tolerant mutants, the cells, were inoculated into acid RCVBH medium (pH 4.5), with the WT strain used as control. Among the 350 mutants, 9 mutants stably produced hydrogen in acid RCVBH medium (pH 4.5) (Table 1). The other mutants and the WT strain did not produce detectable hydrogen. The mutant strain TH-102 produced the highest hydrogen yield in the acid medium. These
results showed that the mutant strain TH-102 has higher resistance to acidic pH than the WT strain. The stability of the mutant strain TH-102 was confirmed by kanamycin resistance after five subcultures in non-selective media. The genomic DNA of the mutant strain TH-102 was purified as the template for PCR. The amplified fragment (about 1699 bp) further confirmed the stability of the generated transposon insertion (Fig. 1).
3.2. Hydrogen production at different initial pHs by TH-102 and the WT strain pH is crucial due to its effects on enzymes function and cell metabolism pathways. The dark-fermentative hydrogen-producing effluent usually has an acidic pH environment, and the hydrogen yield of most of the photosynthetic bacterial strains is low at this acidic pH. Hence, screening of acid tolerant photosynthetic bacteria is very important for successful hydrogen production through integrated dark–photo fermentation. Therefore, the effect of initial pH on hydrogen production was studied at different pH conditions ranging from 4.0 to 9.0. Fig. 2 presents the effects of initial pH on cell growth and total amount of hydrogen produced by the mutant strain TH-102 and the WT strain. It can be noted that both cell growth and hydrogen yield were significantly affected by the initial pH. The acquired mutant strain TH-102 could grow and produce hydrogen at pH 4.0. However, almost no cell growth and hydrogen production of its parent strain R. sulfidophilum P5 was observed at this pH. Similarly, most of the photosynthetic bacterial strains could neither grow nor produce hydrogen at this low pH (Pandey et al., 2012). Under acidic and neutral pH conditions (pH 4.0–7.0), the mutant strain TH-102 produced higher hydrogen content in biogas than the WT strain (P < 0.05); however, under alkali pH conditions (pH 7.5, 8.5 and 9.0), hydrogen content in biogas of the mutant strain TH-102 and WT strain were almost the same. Under acidic pH conditions (pH 4.0–6.5), the mutant strain TH-102 produced higher (486–69%) hydrogen yield than the WT strain (P < 0.05); however, under neutral and weak alkali pH conditions (pH 7.0–8.0), hydrogen yields of the mutant strain TH-102 and WT strain were almost the same. On the other hand, under alkali pH conditions (pH 8.5–9.0), the mutant strain TH-102 produced less hydrogen (46–52%) than the WT strain (P < 0.05). Subsequent research also reported that a photosynthetic bacterium, Rhodopseudomonas faecalis strain RLD-53, evolved different hydrogen content and hydrogen yield under different initial pH condition (Ren et al., 2009). The optimum pH for hydrogen production by the mutant strain TH-102 was observed to be 6.0. The hydrogen yield of TH-102 was approximately 1.68-fold higher than that of the WT strain. This optimum pH value (6.0) for the mutant strain TH-102 is lower than
6
Table 1 Hydrogen-producing ability of the mutants. Strains
Hydrogen content (%)
Hydrogen yield (mol H2/mol acetate)
TH-7 TH-12 TH-65 TH-70 TH-88 TH-102 TH-157 TH-268 TH-291 Wild type
54.53 ± 1.85 41.38 ± 1.08 34.89 ± 2.04 35.23 ± 1.25 37.14 ± 1.32 65.69 ± 2.06 25.64 ± 0.69 30.13 ± 1.42 45.17 ± 0.84 –
0.90 ± 0.08 0.56 ± 0.03 0.93 ± 0.11 0.95 ± 0.12 0.38 ± 0.02 1.08 ± 0.09 0.13 ± 0.02 0.21 ± 0.01 0.51 ± 0.04 –
‘‘–’’ Not detected.
5
4
3
2
1
W M
1699bp
Fig. 1. The stability of inserted transposon Tn7 by PCR amplification. M DNA marker, W: wild-type strain as control, 1–6 six consecutive passages PCR products by the mutant strain TH-102.
257
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
(b) 1600
.7 *
.6
cell dry weight (g/L)
WT TH-102
* *
*
1400
hydrogen production (mL/L)
(a)
*
.5
*
* *
.4
.3 * .2 *
*
1200 * 1000
.1
* 800 600
*
*
400 200
* *
0
0.0 4
5
6
7
8
4
9
5
6
7
initial pH
8
9
initial pH
(c) 100
(d) WT TH-102
* * *
80
*
*
WT TH-102
10
*
* *
8
*
60
*
final pH
hydrogen content (%)
WT TH-102
*
* * *
6 *
40
4
20 4
5
6
7
8
4
9
5
6
7
8
9
initial pH
initial pH
(e) 160 * 140
WT TH-102
* *
120
duration (h)
* 100
80
*
60
40 * 20 4
5
6
7
8
9
initial pH Fig. 2. Effect of initial pH on hydrogen production by the mutant strain TH-102 and the wild-type strain. (a) Cell dry weight, (b) hydrogen production, (c) hydrogen content, (d) final pH, and (e) hydrogen production duration (from begin to the end of hydrogen production) (n = 3, Error bars = s.e.m.).
that previously reported for hydrogen production by its parent strain R. sulfidophilum P5 and most of the photosynthetic bacteria. Most of the photosynthetic bacterial strains produced maximum hydrogen yield at neutral pH, and lower initial pH resulted in a sharp decrease in hydrogen yield, such as R. palustris WP3-5 (Wu et al., 2012), R. gelatinosus M002 (Yang et al., 2012), R. sphaeroides ZX-5 (Zhu et al., 2010). During hydrogen production by the mutant strain TH-102, the initial pH of the culture broth (4.0–9.0) increased to 5.16–10.32.
Subsequent research also reported that the pH increased during the hydrogen production process (Cai et al., 2012; Cai and Wang, 2012). Although both TH-102 and the WT strain increased the pH of the acidic culture broth during hydrogen production, the mutant strain exhibited greater ability to increase pH at a lower pH environment than the WT strain. This is likely a side effect of its ability to substrates uptake, H+ uptake, alkali secretion (Gabrielyan and Trchounian, 2009, 2012), or with formation of products such as polyhydroxybutyrate (Hustede et al., 1993; Kim et al., 2006) at
258
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
conditions restrictive to the WT. Thus, this feature was found to facilitate TH-102 to increase the low pH of the dark-fermentation effluent and simultaneously produce hydrogen. The mutant strain TH-102 exhibited increased cell growth and hydrogen production at acidic pH than the WT strain. To our knowledge, no similar results have been reported for other hydrogen-producing photosynthetic bacterial strains. Nevertheless, the mechanism of acidic pH resistance of the mutant strain TH-102 requires further study.
and hydrogen production rate when the temperature was above 35 °C, irrespective of the illumination wavelength (520 or 590 nm). In addition, He et al. (2006) demonstrated that the culture temperature significantly influenced bacteria growth, hydrogen production rate, and substrate conversion efficiency. The optimal growth temperature for photo-hydrogen production by R. capsulatus strains JP91 and IR3 was noted to be 30 °C. When the temperature was increased to 34 °C, the hydrogen yield of JP91 and IR3 decreased to 24.03% and 33.43%, respectively.
3.3. Hydrogen production at different culture temperatures by TH-102 and the WT strain
3.4. Continuous hydrogen production in semi-batch culture Although the acid tolerant mutant strain TH-102 was found to be a suitable strain for hydrogen production, there are still many practical issues with regard to the use of photosynthetic bacteria for producing hydrogen. As batch process involves regular down time and non-steady-state conditions, continuous process is potentially more attractive. In the present study, 500 mL of working volume was used to examine continuous hydrogen production in semi-batch culture. After 5 days of batch culture, the reactors were fed daily. Fig. 3 illustrates the hydrogen production of the mutant strain TH-102 and the WT strain. The mutant strain TH-102 successfully produced hydrogen, reaching a maximum hydrogen production rate of 14.35 ± 0.38 mL H2/L h on day 4. Subsequently the hydrogen production rate stabilized between 9.02 ± 0.38 and 12.75 ± 0.71 mL H2/L h. In semi-batch operation, the total hydrogen yield and average production rate of the mutant strain TH-102 were 2.16 ± 0.10 mol H2/molacetate and 10.06 ± 0.47 mL H2/L h, respec-
400
hydrogen production (mL/L d)
Some studies have demonstrated that induction of an acid tolerance response in bacteria could provide cross-protection against a wide variety of other stressors (Farber and Pagotto, 1992; Leyer and Johnson, 1993). In most of the dark-fermentative hydrogen production processes, a temperature range of 35–37 °C is commonly employed. However, the optimum hydrogen-producing temperature for most of the photosynthetic bacteria is about 30 °C (Cai and Wang, 2012; He et al., 2006; Wang et al., 2010). For the successful integration of dark and photo fermentation processes for hydrogen production, the hydrogen production performance of the mutant strain TH-102 and the WT strain were examined at a temperature range of 30–40 °C. The initial pH was set to 5.5, because it is the usual pH of the dark-fermentative hydrogen-producing effluent. Table 2 illustrates the hydrogen production of the mutant strain TH-102 and the WT strain at different culture temperatures. The optimum temperature for hydrogen production by the mutant strain TH-102 and the WT strain was found to be 30 °C. When the temperature was increased to 35 °C, the hydrogen yield of the mutant strain TH-102 was almost the same as that at 30 °C (P > 0.05); however, the WT strain exhibited a sharp decrease in the hydrogen yield (P < 0.05). Similarly, Farber and Pagotto (1992) demonstrated increased heat resistance in acid-adapted Listeria monocytogenes. On the other hand, when the temperature was further increased from 35 to 40 °C, the decrease in the hydrogen yield of the mutant strain TH-102 was much lesser than that of the WT strain. These findings revealed that the mutant strain TH-102 was more tolerant to heat than the WT strain. Numerous studies have reported on hydrogen production at different culture temperatures. The mutant strain TH-102 exhibited robust hydrogen production at 35 and 37 °C, making it a suitable candidate for use in integrated dark–photo fermentation process for hydrogen production. Most of the photosynthetic bacteria have been observed to produce low hydrogen yield at 35 and 37 °C; however, at lower temperatures, they have been noted to exhibit robust hydrogen production. Wang et al. (2010) found that the optimal growth and hydrogen production temperature for R. palustris CQK 01 is 30 °C, and observed a sharp decline in hydrogen yield
300
200
continuous
batch 100
WT TH-102 0 0
5
10
15
20
25
30
time (d) Fig. 3. Hydrogen production processes of the mutant strain TH-102 and the wildtype strain P5 in semi-batch operation mode (n = 3, Error bars = s.e.m.).
Table 2 Effect of culture temperature on hydrogen production by the mutant strain TH-102 and the wild-type strain. Temperature (°C) 30 35 37 40
WT TH-102 WT TH-102 WT TH-102 WT TH-102
Cell dry weight (g/L)
Hydrogen production (mL/L)
Hydrogen content in biogas (%)
Final pH
Duration (h)
0.2528 ± 0.0092 0.5281 ± 0.0048* 0.2600 ± 0.0318 0.5425 ± 0.0018* 0.2085 ± 0.0092 0.5001 ± 0.0019* 0.211 ± 0.008 0.1320 ± 0.0149*
170 ± 12 1004 ± 21* 78 ± 6 976 ± 16* 42 ± 4 434 ± 25* – 55 ± 4*
52.20 ± 2.91 82.75 ± 2.11* 45.31 ± 1.51 73.20 ± 1.48* 29.65 ± 3.54 52.81 ± 2.22* – 28.30 ± 2.61*
6.46 ± 0.16 7.21 ± 0.09* 6.66 ± 0.13 7.28 ± 0.06* 6.03 ± 0.27 6.55 ± 0.28 5.72 ± 0.08 6.15 ± 0.15*
48 ± 4 122 ± 9 28 ± 4 97 ± 7 20 ± 2 80 ± 8 – 25 ± 1
Duration: from begin to the end of hydrogen production. ‘‘–’’ Not detected. * T test, P < 0.05.
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
tively, which were about 17.32 and 15.37-fold higher than those of the WT strain (P < 0.05), respectively. These results indicate that the acquired mutant strain TH-102 has high acid and temperature tolerance, making it a suitable starting strain for hydrogen production, especially in integrated fermentative and photosynthetic hydrogen production process in the future. Although in recent years, continuous hydrogen production from various photosynthetic bacteria has been documented (Wang et al., 2010), there has been no report on hydrogen production at low pH (pH 5.5) and high temperature (35 °C), which reflects the conditions of the dark-fermentative hydrogen-producing effluent. 4. Conclusion In conclusion, an acid tolerant mutant TH-102 was acquired from the transposon mutation bank, which exhibited increased hydrogen production than the WT strain at acidic pH and high temperature. In addition, hydrogen production by the mutant strain TH-102 was more stable and highly efficient than its parental WT strain in the semi-batch culture process. Thus, the mutant strain TH-102 with features of acid and temperature tolerance is a potential candidate for use in cost-effective hydrogen production processes, especially in those employing both dark-fermentative and photosynthetic bacteria. Acknowledgements The authors acknowledge the financial support by the Doctoral Fund of Ministry of Education of China (20121208110001), the Key Natural Science Foundation of Tianjin of China (12JCZDJC22200), and the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry (Tianjin University of Science & Technology) (No. 201207). References Anam, K., Habibi, M.S., Harwati, T.U., Susilaningsih, D., 2012. Photofermentative hydrogen production using Rhodobium marinum from bagasse and soy sauce wastewater. Int. J. Hydrogen Energy 37, 15436–15442. Asada, Y., Tokumoto, M., Aihara, Y., Oku, M., Ishimi, K., Wakayama, T., Miyake, J., Tomiyama, M., Kohno, H., 2006. Hydrogen production by co-cultures of Lactobacillus and a photosynthetic bacterium, Rhodobacter sphaeroides RV. Int. J. Hydrogen Energy 31, 1509–1513. Cai, J., Wang, G., 2012. Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond. Int. J. Hydrogen Energy 37, 15070–15080. Cai, J., Wang, G., 2013. Screening and hydrogen-producing characters of a highly efficient H2-producing mutant of Rhodovulum sulfidophilum P5. Bioresour. Technol. 142, 18–25. Cai, J., Wang, G., Pan, G., 2012. Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort. Int. J. Hydrogen Energy 37, 4057–4067. Chen, C.Y., Yang, M.H., Yeh, K.L., Liu, C.H., Chang, J.S., 2008. Biohydrogen production using sequential two-stage dark and photo fermentation processes. Int. J. Hydrogen Energy 33, 4755–4762. Farber, J.M., Pagotto, F., 1992. The effect of acid shock on the heat resistance of Listeria monocytogenes. Lett. Appl. Microbiol. 15, 197–201. Gabrielyan, L., Trchounian, A., 2009. Relationship between molecular hydrogen production, proton transport and the F(0)F(1)-ATPase activity in Rhodobacter sphaeroides strains from mineral springs. Int. J. Hydrogen Energy 34, 2567– 2572. Gabrielyan, L., Trchounian, A., 2012. Concentration dependent glycine effect on the photosynthetic growth and bio-hydrogen production by Rhodobacter sphaeroides from mineral springs. Biomass Bioenergy 36, 333–338. Hallenbeck, P.C., Abo-Hashesh, M., Ghosh, D., 2012. Strategies for improving biological hydrogen production. Bioresour. Technol. 110, 1–9.
259
He, D., Bultel, Y., Magnin, J.P., Willison, J.C., 2006. Kinetic analysis of photosynthetic growth and photohydrogen production of two strains of Rhodobacter capsulatus. Enzyme Microb. Technol. 38, 253–259. Hustede, E., Steinbüchel, A., Schlegel, H.G., 1993. Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria. Appl. Microbiol. Biotechnol. 39, 87–93. Karadag, D., Puhakka, J.A., 2010. Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control. Int. J. Hydrogen Energy 35, 10245–10251. Keskin, T., Abo-Hashesh, M., Hallenbeck, P.C., 2011. Photofermentative hydrogen production from wastes. Bioresour. Technol. 102, 8557–8568. Kim, E.J., Kim, J.S., Kim, M.S., Lee, J.K., 2006a. Effect of changes in the level of light harvesting complexes of Rhodobacter sphaeroides on the photoheterotrophic production of hydrogen. Int. J. Hydrogen Energy 31, 531–538. Kim, M.S., Baek, J.S., Yun, Y.S., Jun Sim, S., Park, S., Kim, S.C., 2006b. Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: anaerobic conversion and photosynthetic fermentation. Int. J. Hydrogen Energy 31, 812–816. Lee, H.-S., Vermaas, W.F.J., Rittmann, B.E., 2010. Biological hydrogen production: prospects and challenges. Trends Biotechnol. 28, 262–271. Leyer, G.J., Johnson, E.A., 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microb. 59, 1842–1847. Liu, B.F., Ren, N.Q., Xing, D.F., Ding, J., Zheng, G.X., Guo, W.Q., Xu, J.F., Xie, G.J., 2009. Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49. Bioresour. Technol. 100, 2719–2723. Ljunggren, M., Willquist, K., Zacchi, G., van Niel, E.W.J., 2011. A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus. Biotechnol. Biofuels 4. Luo, G., Xie, L., Zou, Z., Zhou, Q., Wang, J.Y., 2010. Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: effects of temperature and pH. Appl. Energy 87, 3710–3717. Ma, C., Guo, L., Yang, H., 2012. Improved photo – Hydrogen production by transposon mutant of Rhodobacter capsulatus with reduced pigment. Int. J. Hydrogen Energy 37, 8282–8287. Masset, J., Calusinska, M., Hamilton, C., Hiligsmann, S., Joris, B., Wilmotte, A., Thonart, P., 2012. Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp. Biotechnol. Biofuels 5. Matsunaga, T., Hatano, T., Yamada, A., Matsumoto, M., 2000. Microaerobic hydrogen production by photosynthetic bacteria in a double-phase photobioreactor. Biotechnol. Bioeng. 68, 647–651. Nath, K., Das, D., 2009. Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process. Int. J. Hydrogen Energy 34, 7497–7501. Pandey, A., Srivastava, N., Sinha, P., 2012. Optimization of hydrogen production by Rhodobacter sphaeroides NMBL-01. Biomass Bioenergy 37, 251–256. Patel, S.K.S., Kumar, P., Kalia, V.C., 2012. Enhancing biological hydrogen production through complementary microbial metabolisms. Int. J. Hydrogen Energy 37, 10590–10603. Ren, N.Q., Liu, B.F., Ding, J., Xie, G.J., 2009. Hydrogen production with R. faecalis RLD53 isolated from freshwater pond sludge. Bioresour. Technol. 100, 484–487. Sigurbjornsdottir, M.A., Orlygsson, J., 2012. Combined hydrogen and ethanol production from sugars and lignocellulosic biomass by Thermoanaerobacterium AK54, isolated from hot spring. Appl. Energy 97, 785– 791. Vanzin, G., Yu, J., Smolinski, S., Tek, V., Pennington, G., Maness, P.C., 2010. Characterization of genes responsible for the co-Linked hydrogen production pathway in Rubrivivax gelatinosus. Appl. Environ. Microb. 76, 3715–3722. Wang, B., Wan, W., Wang, J., 2008. Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production. Int. J. Hydrogen Energy 33, 7013–7019. Wang, Y.Z., Liao, Q., Zhu, X., Tian, X., Zhang, C., 2010. Characteristics of hydrogen production and substrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor. Bioresour. Technol. 101, 4034–4041. Wu, S.C., Liou, S.Z., Lee, C.M., 2012. Correlation between bio-hydrogen production and polyhydroxybutyrate (PHB) synthesis by Rhodopseudomonas palustris WP35. Bioresour. Technol. 113, 44–50. Yang, H., Wang, X., Zhang, L., Guo, L., 2012. Enhanced hydrogen production performance of Rubrivivax gelatinosus M002 using mixed carbon sources. Int. J. Hydrogen Energy 37, 13296–13303. Zhu, Z.N., Shi, J.P., Zhou, Z.H., Hu, F.X., Bao, J., 2010. Photo-fermentation of Rhodobacter sphaeroides for hydrogen production using lignocellulose-derived organic acids. Process Biochem. 45, 1894–1898.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Photo-biological hydrogen production by an acid tolerant mutant of Rhodovulum sulfidophilum P5 generated by transposon mutagenesis Jinling Cai a,⇑, Guangce Wang a,b,⇑ a b
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, PR China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong, PR China
h i g h l i g h t s Transposon mutagenesis was used to enhance H2 yield of a photosynthetic bacterium. A transposon mutagenesis library of Rhodovulum sulfidophilum P5 was constructed. Mutant TH-102 had higher aciduric and temperature resistant ability. TH-102 can produce H2 at dark fermentation effluent environment (pH 5.5 and 35 °C). In continuous culture, H2 yield and rate were 17 and 15-fold higher than the WT.
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 10 December 2013 Accepted 12 December 2013 Available online 22 December 2013 Keywords: Hydrogen production Photosynthetic bacteria Acid tolerant Transposon Mutation
a b s t r a c t Most of the photosynthetic bacterial strains exhibit optimum hydrogen production at neutral initial pH, and lower initial pH resulted in a sharp decrease in hydrogen yield. Thus, screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. To obtain acid tolerant mutants, a Tn7based transposon was randomly inserted into the genomic DNA of Rhodovulum sulfidophilum P5. An acid tolerant mutant strain TH-102 exhibited increased hydrogen production in acidic environment (pH 4.5– 6.5) and at higher temperatures (35 and 37 °C) than the wild-type strain. At pH 5.5 and 35 °C, the mutant strain TH-102 continuously produced hydrogen. The hydrogen yield and average rate were 2.16 ± 0.10 mol/mol acetate and 10.06 ± 0.47 mL/L h, which was about 17.32 and 15.37-fold higher than that of the wild-type strain, respectively. This acid- and temperature-tolerant mutant strain TH-102 could be used in a cost-effective hydrogen production process employing both dark fermentative and photosynthetic bacteria. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Due to the limited availability of traditional energy from a nonrenewable reservoir and as a result of ever-growing energy demand, there is an increasing interest in the search for renewable energy sources to meet the current and future energy requirements. Numerous alternative and renewable energy resources have been explored. Among them, hydrogen is an attractive potential alternative energy source due to its non-polluting and environmental-friendly nature, and currently, sustainable biological hydrogen production is under active investigation. The low yield and production rate are still major barriers for commercial biological production of hydrogen. It has been
⇑ Corresponding authors at: Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, PR China. Tel./fax: +86 022 60601305 (G. Wang). E-mail addresses: [email protected] (J. Cai), [email protected] (G. Wang). 0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.057
estimated that a hydrogen yield of 8 mol H2/mol glucose would be sufficient for practical production (Keskin et al., 2011). However, the hydrogen yield of all the principal biological hydrogen production methods such as direct and indirect biophotolysis, photo fermentation, and dark fermentation, is relatively below this benchmark. In some studies, integration of dark and photo fermentations has been reported to produce yields of up to 7.1 mol H2/mol hexose (Asada et al., 2006; Chen et al., 2008) (even up to 8.3 mol H2/mol hexose (Kim et al., 2006)). Thus, further investigations are required for the development of these systems for practical purposes. The combined use of dark-fermentative bacteria and photosynthetic bacteria, which is a promising hydrogen production approach, can markedly enhance hydrogen yield. Most of the dark-fermentative bacteria can produce hydrogen at high rate, but cannot degrade organic compounds completely due to thermodynamic limitations. The maximum theoretical hydrogen yield of dark fermentation is relatively low, ranging from 2 to 4 mol/mol hexose according to the composition of the organic acids produced.
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
As a result, the dark-fermentative hydrogen-producing effluent primarily contains organic acids (mainly acetate and butyrate) and alcohols (Lee et al., 2010), mostly in their undissociated forms, which can cross the cell membrane at a low pH and can adversely affect hydrogen production (Wang et al., 2008). Furthermore, the presence of organic acids and alcohols in the residual medium poses a disposal issue. However, the organic acids could be further metabolized to produce additional hydrogen by photosynthetic bacteria under photoheterotrophic conditions, which increases the theoretical hydrogen yield from 4 to 12 mol H2/mol glucose and reduces the organic content of the final residual waste. Several studies have reported that the combined use of both dark-fermentative and photosynthetic bacteria could achieve higher hydrogen yields from various substrates, when compared with the hydrogen yields obtained using either of these bacteria (Lee et al., 2010; Liu et al., 2009; Patel et al., 2012). Thus, the combined use of darkfermentative and photosynthetic bacteria may overcome individual limitations and exhibit greater advantages. Low pH of the dark-fermentative effluent inhibits hydrogen production and even growth of the photosynthetic bacteria, which is one of the major barriers for the stable operation of the integrated process. The pH for dark fermentative effluent and optimum pH for hydrogen production are in the acidic range of 4.5–5.5 (Karadag and Pahuakka, 2010; Luo et al., 2010; Masset et al., 2012; Sigurbjornsdottir and Orlygsson, 2012). However, the optimum pH for hydrogen production by most of the photosynthetic bacteria is neutral (Nath and Das, 2009; Wu et al., 2012; Yang et al., 2012; Zhu et al., 2010) to weak alkaline (Cai et al., 2012; Cai and Wang, 2012; Pandey et al., 2012). It has been observed to be necessary to control the pH of the fermentation effluent for further production of hydrogen by photosynthetic bacteria (Ljunggren et al., 2011). However, controlling the pH of the dark fermentation process requires large amounts of alkaline solution, which not only presents an economic burden, but also increases the concentration of salts in the treated effluent, requiring adoption of an efficient way to reduce the amount of alkaline solution. Hence, it is necessary to achieve acid tolerant remediation of fermentation effluent, which could reduce alkali consumption, as well as increase the hydrogen yield. Screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. These bacteria are not only acid tolerant, adapting to acid stress and alleviating organic acid inhibition, but are also beneficial for hydrogen production as well as for the integration of dark and photo fermentations. Although the traditional isolation strategy for screening acid-tolerant photosynthetic bacteria for hydrogen production is effective, it is time-consuming. Some powerful genetic engineering approaches could be employed to improve the hydrogen yield (Hallenbeck et al., 2012; Kim et al., 2006). Among them, transposon technology, carrying antibioticresistance genes, is an excellent indispensable tool in bacterial genetics, especially for those bacteria whose genetic system has not yet been developed (Ma et al., 2012; Cai and Wang, 2013). Transposon mutagenesis is an important genetic tool for the creation and characterization of insertion mutants, and has been used to carry out insertional mutagenesis screening of mutants of photosynthetic bacteria which could produce high hydrogen yield, such as Rhodovulum gelatinosus (Vanzin et al., 2010), Rhodovulum capsulatus (Ma et al., 2012), and R. sulfidophilum P5 (Cai and Wang, 2013). Furthermore, the use of transposon mutagenesis technology to create a mutant bank of R. sulfidophilum P5 could significantly contribute to enhancing its hydrogen production as well as our understanding of the mechanism of hydrogen production. To date, biological hydrogen production by photosynthetic bacteria in fresh conditions has been well developed. Hydrogen production by marine photosynthetic bacteria from marine wastewater has been attracting increasing attention. Some studies
255
have reported hydrogen production by marine photosynthetic bacteria, such as Rhodobium marinum (Anam et al., 2012), R. sulfidophilum P5 (Cai and Wang, 2012), and Rhodovulum spp. (Matsunaga et al., 2000), as well as marine mixed phototrophic bacterial consort (Cai et al., 2012). However, most of the investigations have been mainly focused on optimizing the basic parameters, including the operating conditions, substrate selection, immobilization of photosynthetic bacteria cells for a higher retention time, etc. Only a few studies have attempted to improve the hydrogen yield of photosynthetic bacteria through molecular biology methods (Ma et al., 2012). Thus, the main purpose of the present study was to screen acid-tolerant photosynthetic bacteria producing high hydrogen yield, which could be used in the integrated dark–photo fermentation process for hydrogen production. 2. Methods 2.1. Bacterial strain and culture medium The transposon library of R. sulfidophilum P5 was created according the forward research (Cai and Wang, 2013). Transposition was performed using the GPS mutagenesis system (New England Biolabs Catalog NO E7101S), according to the manufacturer’s instructions. The transprimer donor used was pGPS3, which carries kanamycin resistance. The acquired mutants were stored at 80 °C in 15% glycerol. R. sulfidophilum strains were grown on RCVBH medium (Cai and Wang, 2012). The RCVBH medium contained the following: 20 mmol/L acetate, 10 mmol/L glutamate, 20 g/L NaCl, 75 mg/L CaCl2 2H2O, 120 mg/L MgSO4 7H2O, 10 mmol/L KPO4 buffer, 1 mg/L thiamine hydrochloride, 20 mg/L sodium ethylenediaminetetraacetic acid, and 1 mL/L trace element solution (per 100 mL of deionized water (dH2O): 280 mg H3BO3, 75.2 mg NaMoO4 2H2O, 159.2 mg MnSO4 H2O, 3H2O, 4 mg Cu(NO3)2, and 24 mg ZnSO4 7H2O). For basic growth, the pH was adjusted to 8.0. 2.2. Screen and characterization of acid tolerant mutant To verify the hydrogen production ability of the mutants, hydrogen production profiles of different mutant strains were grown under acidic condition (pH 4.5) using 50 mL anaerobic tube (20 mL working volume). Among all the mutants, only 9 mutants could produce hydrogen under acidic condition (pH 4.5). These nine acid tolerant mutant strains could evolve hydrogen were further analyzed the nature of hydrogen production. A total of 50 mL of pre-cultured cells (OD660 = 0.8–1.0) were harvested by centrifugation and inoculated into 500 mL of RCVBH medium. Argon gas was purged into the bioreactors to create anaerobic conditions. The bioreactors were stirred at 150 rpm at 30 °C. The light intensity was 100 lmol photons/m2 s. For further analysis of the hydrogen production nature by the acid tolerant mutant strain TH-102, two series of test batches (of 500-mL working volume) were cultured under anaerobic conditions in RCVBH medium, with the WT strain used as the control. For the initial pH tests, the pH was varied from 4.0 to 9.0. For the culture temperature test, the temperatures ranged from 30 to 40 °C. For semi-batch hydrogen production, the mutant strain TH-102 and the WT strain in exponential growth phase were harvested and inoculated into 500-mL culture medium. Initially, the reactors were operated in batch mode for 5 days to accumulate biomass for continuous operation. Thereafter, 125 mL of the exhausted medium was withdrawn and replaced with fresh medium. The reactor was operated under the same conditions as described
256
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
earlier. The culture temperature was 35 °C and the initial pH was 5.5, reflecting the environment of dark-fermentative hydrogenproducing effluent. 2.3. Stability of acid tolerant mutants The stability of the mutant strain TH-102 was by preparing five subcultures in non-selective media, and then inoculating the cells into kanamycin media (50 lL/mL kanamycin sulfate). The transposon fragment was confirmed by PCR using primers derived from transposon sequence (P1: 50 -TTAAGGATTATTTAGGGAAG-30 ; P2: 50 -ACAATAAAGTCTTAAACTAG-30 ). The PCR program was as follows: 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. After completion of 30 cycles, a 10-min extension was run at 72 °C. The identity of the generated transposon fragment was further verified mutation stability of the mutant strain TH-102. 2.4. Analytical methods When hydrogen production ceased for each tests, hydrogen and cell dry weight were analyzed. Hydrogen was analyzed by gas chromatography (GC-2010, Tengzhou, China), equipped with a 2-m stainless steel column packed with Porapak DMCS (60/80 mesh) and a thermal conductivity detector. The operating procedure was according to that reported in a previous study (Cai and Wang, 2012). The cell dry weight was determined as described previously (Cai et al., 2012). 2.5. Statistical analysis All the results were presented as mean ± standard deviation (SD) values of the triplicates for each experiment. Statistical analyses were performed using the IBM SPSS Statistics 19 package (IBM Co., Armonk, New York, USA). T-tests (a = 0.05) were used to determined whether significant differences were exist between various treatment groups. 3. Results and discussion 3.1. Acid tolerant mutants screen About 350 mutant colonies were acquired from forward transposon mutagenesis system (Cai and Wang, 2013). To screen acid tolerant mutants, the cells, were inoculated into acid RCVBH medium (pH 4.5), with the WT strain used as control. Among the 350 mutants, 9 mutants stably produced hydrogen in acid RCVBH medium (pH 4.5) (Table 1). The other mutants and the WT strain did not produce detectable hydrogen. The mutant strain TH-102 produced the highest hydrogen yield in the acid medium. These
results showed that the mutant strain TH-102 has higher resistance to acidic pH than the WT strain. The stability of the mutant strain TH-102 was confirmed by kanamycin resistance after five subcultures in non-selective media. The genomic DNA of the mutant strain TH-102 was purified as the template for PCR. The amplified fragment (about 1699 bp) further confirmed the stability of the generated transposon insertion (Fig. 1).
3.2. Hydrogen production at different initial pHs by TH-102 and the WT strain pH is crucial due to its effects on enzymes function and cell metabolism pathways. The dark-fermentative hydrogen-producing effluent usually has an acidic pH environment, and the hydrogen yield of most of the photosynthetic bacterial strains is low at this acidic pH. Hence, screening of acid tolerant photosynthetic bacteria is very important for successful hydrogen production through integrated dark–photo fermentation. Therefore, the effect of initial pH on hydrogen production was studied at different pH conditions ranging from 4.0 to 9.0. Fig. 2 presents the effects of initial pH on cell growth and total amount of hydrogen produced by the mutant strain TH-102 and the WT strain. It can be noted that both cell growth and hydrogen yield were significantly affected by the initial pH. The acquired mutant strain TH-102 could grow and produce hydrogen at pH 4.0. However, almost no cell growth and hydrogen production of its parent strain R. sulfidophilum P5 was observed at this pH. Similarly, most of the photosynthetic bacterial strains could neither grow nor produce hydrogen at this low pH (Pandey et al., 2012). Under acidic and neutral pH conditions (pH 4.0–7.0), the mutant strain TH-102 produced higher hydrogen content in biogas than the WT strain (P < 0.05); however, under alkali pH conditions (pH 7.5, 8.5 and 9.0), hydrogen content in biogas of the mutant strain TH-102 and WT strain were almost the same. Under acidic pH conditions (pH 4.0–6.5), the mutant strain TH-102 produced higher (486–69%) hydrogen yield than the WT strain (P < 0.05); however, under neutral and weak alkali pH conditions (pH 7.0–8.0), hydrogen yields of the mutant strain TH-102 and WT strain were almost the same. On the other hand, under alkali pH conditions (pH 8.5–9.0), the mutant strain TH-102 produced less hydrogen (46–52%) than the WT strain (P < 0.05). Subsequent research also reported that a photosynthetic bacterium, Rhodopseudomonas faecalis strain RLD-53, evolved different hydrogen content and hydrogen yield under different initial pH condition (Ren et al., 2009). The optimum pH for hydrogen production by the mutant strain TH-102 was observed to be 6.0. The hydrogen yield of TH-102 was approximately 1.68-fold higher than that of the WT strain. This optimum pH value (6.0) for the mutant strain TH-102 is lower than
6
Table 1 Hydrogen-producing ability of the mutants. Strains
Hydrogen content (%)
Hydrogen yield (mol H2/mol acetate)
TH-7 TH-12 TH-65 TH-70 TH-88 TH-102 TH-157 TH-268 TH-291 Wild type
54.53 ± 1.85 41.38 ± 1.08 34.89 ± 2.04 35.23 ± 1.25 37.14 ± 1.32 65.69 ± 2.06 25.64 ± 0.69 30.13 ± 1.42 45.17 ± 0.84 –
0.90 ± 0.08 0.56 ± 0.03 0.93 ± 0.11 0.95 ± 0.12 0.38 ± 0.02 1.08 ± 0.09 0.13 ± 0.02 0.21 ± 0.01 0.51 ± 0.04 –
‘‘–’’ Not detected.
5
4
3
2
1
W M
1699bp
Fig. 1. The stability of inserted transposon Tn7 by PCR amplification. M DNA marker, W: wild-type strain as control, 1–6 six consecutive passages PCR products by the mutant strain TH-102.
257
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
(b) 1600
.7 *
.6
cell dry weight (g/L)
WT TH-102
* *
*
1400
hydrogen production (mL/L)
(a)
*
.5
*
* *
.4
.3 * .2 *
*
1200 * 1000
.1
* 800 600
*
*
400 200
* *
0
0.0 4
5
6
7
8
4
9
5
6
7
initial pH
8
9
initial pH
(c) 100
(d) WT TH-102
* * *
80
*
*
WT TH-102
10
*
* *
8
*
60
*
final pH
hydrogen content (%)
WT TH-102
*
* * *
6 *
40
4
20 4
5
6
7
8
4
9
5
6
7
8
9
initial pH
initial pH
(e) 160 * 140
WT TH-102
* *
120
duration (h)
* 100
80
*
60
40 * 20 4
5
6
7
8
9
initial pH Fig. 2. Effect of initial pH on hydrogen production by the mutant strain TH-102 and the wild-type strain. (a) Cell dry weight, (b) hydrogen production, (c) hydrogen content, (d) final pH, and (e) hydrogen production duration (from begin to the end of hydrogen production) (n = 3, Error bars = s.e.m.).
that previously reported for hydrogen production by its parent strain R. sulfidophilum P5 and most of the photosynthetic bacteria. Most of the photosynthetic bacterial strains produced maximum hydrogen yield at neutral pH, and lower initial pH resulted in a sharp decrease in hydrogen yield, such as R. palustris WP3-5 (Wu et al., 2012), R. gelatinosus M002 (Yang et al., 2012), R. sphaeroides ZX-5 (Zhu et al., 2010). During hydrogen production by the mutant strain TH-102, the initial pH of the culture broth (4.0–9.0) increased to 5.16–10.32.
Subsequent research also reported that the pH increased during the hydrogen production process (Cai et al., 2012; Cai and Wang, 2012). Although both TH-102 and the WT strain increased the pH of the acidic culture broth during hydrogen production, the mutant strain exhibited greater ability to increase pH at a lower pH environment than the WT strain. This is likely a side effect of its ability to substrates uptake, H+ uptake, alkali secretion (Gabrielyan and Trchounian, 2009, 2012), or with formation of products such as polyhydroxybutyrate (Hustede et al., 1993; Kim et al., 2006) at
258
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
conditions restrictive to the WT. Thus, this feature was found to facilitate TH-102 to increase the low pH of the dark-fermentation effluent and simultaneously produce hydrogen. The mutant strain TH-102 exhibited increased cell growth and hydrogen production at acidic pH than the WT strain. To our knowledge, no similar results have been reported for other hydrogen-producing photosynthetic bacterial strains. Nevertheless, the mechanism of acidic pH resistance of the mutant strain TH-102 requires further study.
and hydrogen production rate when the temperature was above 35 °C, irrespective of the illumination wavelength (520 or 590 nm). In addition, He et al. (2006) demonstrated that the culture temperature significantly influenced bacteria growth, hydrogen production rate, and substrate conversion efficiency. The optimal growth temperature for photo-hydrogen production by R. capsulatus strains JP91 and IR3 was noted to be 30 °C. When the temperature was increased to 34 °C, the hydrogen yield of JP91 and IR3 decreased to 24.03% and 33.43%, respectively.
3.3. Hydrogen production at different culture temperatures by TH-102 and the WT strain
3.4. Continuous hydrogen production in semi-batch culture Although the acid tolerant mutant strain TH-102 was found to be a suitable strain for hydrogen production, there are still many practical issues with regard to the use of photosynthetic bacteria for producing hydrogen. As batch process involves regular down time and non-steady-state conditions, continuous process is potentially more attractive. In the present study, 500 mL of working volume was used to examine continuous hydrogen production in semi-batch culture. After 5 days of batch culture, the reactors were fed daily. Fig. 3 illustrates the hydrogen production of the mutant strain TH-102 and the WT strain. The mutant strain TH-102 successfully produced hydrogen, reaching a maximum hydrogen production rate of 14.35 ± 0.38 mL H2/L h on day 4. Subsequently the hydrogen production rate stabilized between 9.02 ± 0.38 and 12.75 ± 0.71 mL H2/L h. In semi-batch operation, the total hydrogen yield and average production rate of the mutant strain TH-102 were 2.16 ± 0.10 mol H2/molacetate and 10.06 ± 0.47 mL H2/L h, respec-
400
hydrogen production (mL/L d)
Some studies have demonstrated that induction of an acid tolerance response in bacteria could provide cross-protection against a wide variety of other stressors (Farber and Pagotto, 1992; Leyer and Johnson, 1993). In most of the dark-fermentative hydrogen production processes, a temperature range of 35–37 °C is commonly employed. However, the optimum hydrogen-producing temperature for most of the photosynthetic bacteria is about 30 °C (Cai and Wang, 2012; He et al., 2006; Wang et al., 2010). For the successful integration of dark and photo fermentation processes for hydrogen production, the hydrogen production performance of the mutant strain TH-102 and the WT strain were examined at a temperature range of 30–40 °C. The initial pH was set to 5.5, because it is the usual pH of the dark-fermentative hydrogen-producing effluent. Table 2 illustrates the hydrogen production of the mutant strain TH-102 and the WT strain at different culture temperatures. The optimum temperature for hydrogen production by the mutant strain TH-102 and the WT strain was found to be 30 °C. When the temperature was increased to 35 °C, the hydrogen yield of the mutant strain TH-102 was almost the same as that at 30 °C (P > 0.05); however, the WT strain exhibited a sharp decrease in the hydrogen yield (P < 0.05). Similarly, Farber and Pagotto (1992) demonstrated increased heat resistance in acid-adapted Listeria monocytogenes. On the other hand, when the temperature was further increased from 35 to 40 °C, the decrease in the hydrogen yield of the mutant strain TH-102 was much lesser than that of the WT strain. These findings revealed that the mutant strain TH-102 was more tolerant to heat than the WT strain. Numerous studies have reported on hydrogen production at different culture temperatures. The mutant strain TH-102 exhibited robust hydrogen production at 35 and 37 °C, making it a suitable candidate for use in integrated dark–photo fermentation process for hydrogen production. Most of the photosynthetic bacteria have been observed to produce low hydrogen yield at 35 and 37 °C; however, at lower temperatures, they have been noted to exhibit robust hydrogen production. Wang et al. (2010) found that the optimal growth and hydrogen production temperature for R. palustris CQK 01 is 30 °C, and observed a sharp decline in hydrogen yield
300
200
continuous
batch 100
WT TH-102 0 0
5
10
15
20
25
30
time (d) Fig. 3. Hydrogen production processes of the mutant strain TH-102 and the wildtype strain P5 in semi-batch operation mode (n = 3, Error bars = s.e.m.).
Table 2 Effect of culture temperature on hydrogen production by the mutant strain TH-102 and the wild-type strain. Temperature (°C) 30 35 37 40
WT TH-102 WT TH-102 WT TH-102 WT TH-102
Cell dry weight (g/L)
Hydrogen production (mL/L)
Hydrogen content in biogas (%)
Final pH
Duration (h)
0.2528 ± 0.0092 0.5281 ± 0.0048* 0.2600 ± 0.0318 0.5425 ± 0.0018* 0.2085 ± 0.0092 0.5001 ± 0.0019* 0.211 ± 0.008 0.1320 ± 0.0149*
170 ± 12 1004 ± 21* 78 ± 6 976 ± 16* 42 ± 4 434 ± 25* – 55 ± 4*
52.20 ± 2.91 82.75 ± 2.11* 45.31 ± 1.51 73.20 ± 1.48* 29.65 ± 3.54 52.81 ± 2.22* – 28.30 ± 2.61*
6.46 ± 0.16 7.21 ± 0.09* 6.66 ± 0.13 7.28 ± 0.06* 6.03 ± 0.27 6.55 ± 0.28 5.72 ± 0.08 6.15 ± 0.15*
48 ± 4 122 ± 9 28 ± 4 97 ± 7 20 ± 2 80 ± 8 – 25 ± 1
Duration: from begin to the end of hydrogen production. ‘‘–’’ Not detected. * T test, P < 0.05.
J. Cai, G. Wang / Bioresource Technology 154 (2014) 254–259
tively, which were about 17.32 and 15.37-fold higher than those of the WT strain (P < 0.05), respectively. These results indicate that the acquired mutant strain TH-102 has high acid and temperature tolerance, making it a suitable starting strain for hydrogen production, especially in integrated fermentative and photosynthetic hydrogen production process in the future. Although in recent years, continuous hydrogen production from various photosynthetic bacteria has been documented (Wang et al., 2010), there has been no report on hydrogen production at low pH (pH 5.5) and high temperature (35 °C), which reflects the conditions of the dark-fermentative hydrogen-producing effluent. 4. Conclusion In conclusion, an acid tolerant mutant TH-102 was acquired from the transposon mutation bank, which exhibited increased hydrogen production than the WT strain at acidic pH and high temperature. In addition, hydrogen production by the mutant strain TH-102 was more stable and highly efficient than its parental WT strain in the semi-batch culture process. Thus, the mutant strain TH-102 with features of acid and temperature tolerance is a potential candidate for use in cost-effective hydrogen production processes, especially in those employing both dark-fermentative and photosynthetic bacteria. Acknowledgements The authors acknowledge the financial support by the Doctoral Fund of Ministry of Education of China (20121208110001), the Key Natural Science Foundation of Tianjin of China (12JCZDJC22200), and the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry (Tianjin University of Science & Technology) (No. 201207). References Anam, K., Habibi, M.S., Harwati, T.U., Susilaningsih, D., 2012. Photofermentative hydrogen production using Rhodobium marinum from bagasse and soy sauce wastewater. Int. J. Hydrogen Energy 37, 15436–15442. Asada, Y., Tokumoto, M., Aihara, Y., Oku, M., Ishimi, K., Wakayama, T., Miyake, J., Tomiyama, M., Kohno, H., 2006. Hydrogen production by co-cultures of Lactobacillus and a photosynthetic bacterium, Rhodobacter sphaeroides RV. Int. J. Hydrogen Energy 31, 1509–1513. Cai, J., Wang, G., 2012. Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond. Int. J. Hydrogen Energy 37, 15070–15080. Cai, J., Wang, G., 2013. Screening and hydrogen-producing characters of a highly efficient H2-producing mutant of Rhodovulum sulfidophilum P5. Bioresour. Technol. 142, 18–25. Cai, J., Wang, G., Pan, G., 2012. Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort. Int. J. Hydrogen Energy 37, 4057–4067. Chen, C.Y., Yang, M.H., Yeh, K.L., Liu, C.H., Chang, J.S., 2008. Biohydrogen production using sequential two-stage dark and photo fermentation processes. Int. J. Hydrogen Energy 33, 4755–4762. Farber, J.M., Pagotto, F., 1992. The effect of acid shock on the heat resistance of Listeria monocytogenes. Lett. Appl. Microbiol. 15, 197–201. Gabrielyan, L., Trchounian, A., 2009. Relationship between molecular hydrogen production, proton transport and the F(0)F(1)-ATPase activity in Rhodobacter sphaeroides strains from mineral springs. Int. J. Hydrogen Energy 34, 2567– 2572. Gabrielyan, L., Trchounian, A., 2012. Concentration dependent glycine effect on the photosynthetic growth and bio-hydrogen production by Rhodobacter sphaeroides from mineral springs. Biomass Bioenergy 36, 333–338. Hallenbeck, P.C., Abo-Hashesh, M., Ghosh, D., 2012. Strategies for improving biological hydrogen production. Bioresour. Technol. 110, 1–9.
259
He, D., Bultel, Y., Magnin, J.P., Willison, J.C., 2006. Kinetic analysis of photosynthetic growth and photohydrogen production of two strains of Rhodobacter capsulatus. Enzyme Microb. Technol. 38, 253–259. Hustede, E., Steinbüchel, A., Schlegel, H.G., 1993. Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria. Appl. Microbiol. Biotechnol. 39, 87–93. Karadag, D., Puhakka, J.A., 2010. Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control. Int. J. Hydrogen Energy 35, 10245–10251. Keskin, T., Abo-Hashesh, M., Hallenbeck, P.C., 2011. Photofermentative hydrogen production from wastes. Bioresour. Technol. 102, 8557–8568. Kim, E.J., Kim, J.S., Kim, M.S., Lee, J.K., 2006a. Effect of changes in the level of light harvesting complexes of Rhodobacter sphaeroides on the photoheterotrophic production of hydrogen. Int. J. Hydrogen Energy 31, 531–538. Kim, M.S., Baek, J.S., Yun, Y.S., Jun Sim, S., Park, S., Kim, S.C., 2006b. Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: anaerobic conversion and photosynthetic fermentation. Int. J. Hydrogen Energy 31, 812–816. Lee, H.-S., Vermaas, W.F.J., Rittmann, B.E., 2010. Biological hydrogen production: prospects and challenges. Trends Biotechnol. 28, 262–271. Leyer, G.J., Johnson, E.A., 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microb. 59, 1842–1847. Liu, B.F., Ren, N.Q., Xing, D.F., Ding, J., Zheng, G.X., Guo, W.Q., Xu, J.F., Xie, G.J., 2009. Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49. Bioresour. Technol. 100, 2719–2723. Ljunggren, M., Willquist, K., Zacchi, G., van Niel, E.W.J., 2011. A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus. Biotechnol. Biofuels 4. Luo, G., Xie, L., Zou, Z., Zhou, Q., Wang, J.Y., 2010. Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: effects of temperature and pH. Appl. Energy 87, 3710–3717. Ma, C., Guo, L., Yang, H., 2012. Improved photo – Hydrogen production by transposon mutant of Rhodobacter capsulatus with reduced pigment. Int. J. Hydrogen Energy 37, 8282–8287. Masset, J., Calusinska, M., Hamilton, C., Hiligsmann, S., Joris, B., Wilmotte, A., Thonart, P., 2012. Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp. Biotechnol. Biofuels 5. Matsunaga, T., Hatano, T., Yamada, A., Matsumoto, M., 2000. Microaerobic hydrogen production by photosynthetic bacteria in a double-phase photobioreactor. Biotechnol. Bioeng. 68, 647–651. Nath, K., Das, D., 2009. Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process. Int. J. Hydrogen Energy 34, 7497–7501. Pandey, A., Srivastava, N., Sinha, P., 2012. Optimization of hydrogen production by Rhodobacter sphaeroides NMBL-01. Biomass Bioenergy 37, 251–256. Patel, S.K.S., Kumar, P., Kalia, V.C., 2012. Enhancing biological hydrogen production through complementary microbial metabolisms. Int. J. Hydrogen Energy 37, 10590–10603. Ren, N.Q., Liu, B.F., Ding, J., Xie, G.J., 2009. Hydrogen production with R. faecalis RLD53 isolated from freshwater pond sludge. Bioresour. Technol. 100, 484–487. Sigurbjornsdottir, M.A., Orlygsson, J., 2012. Combined hydrogen and ethanol production from sugars and lignocellulosic biomass by Thermoanaerobacterium AK54, isolated from hot spring. Appl. Energy 97, 785– 791. Vanzin, G., Yu, J., Smolinski, S., Tek, V., Pennington, G., Maness, P.C., 2010. Characterization of genes responsible for the co-Linked hydrogen production pathway in Rubrivivax gelatinosus. Appl. Environ. Microb. 76, 3715–3722. Wang, B., Wan, W., Wang, J., 2008. Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production. Int. J. Hydrogen Energy 33, 7013–7019. Wang, Y.Z., Liao, Q., Zhu, X., Tian, X., Zhang, C., 2010. Characteristics of hydrogen production and substrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor. Bioresour. Technol. 101, 4034–4041. Wu, S.C., Liou, S.Z., Lee, C.M., 2012. Correlation between bio-hydrogen production and polyhydroxybutyrate (PHB) synthesis by Rhodopseudomonas palustris WP35. Bioresour. Technol. 113, 44–50. Yang, H., Wang, X., Zhang, L., Guo, L., 2012. Enhanced hydrogen production performance of Rubrivivax gelatinosus M002 using mixed carbon sources. Int. J. Hydrogen Energy 37, 13296–13303. Zhu, Z.N., Shi, J.P., Zhou, Z.H., Hu, F.X., Bao, J., 2010. Photo-fermentation of Rhodobacter sphaeroides for hydrogen production using lignocellulose-derived organic acids. Process Biochem. 45, 1894–1898.
