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Comparison of Ethanol Fermentation Efficiency: Syngases versus Microorganisms

Feng-Qin Wang1#, Yan-da Zhang a1#, Bing-bing Sun2, Hui Xie1, Da-jiao Yang1, Sen -Yang1, Tian-bao Ren3, Sheng Yang2, An-Dong Song1*

1Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, College of Life Science, Henan Agricultural University, Zhengzhou 450002, P. R. China

2Key Laboratory of Synthetic Biology, Chinese Academy of Sciences, Shanghai 200000, P. R. China

3National Tobacco Cultivation and Physiological and Biochemical Research Centre, Henan Agricultural University, Zhengzhou 450002, P. R. China

#Equally Contributed

*Corresponding Author:
An-Dong Song
Henan Agricultural University
Zhengzhou 450002
Henan, P.R. China

Received date: June 16, 2016; Accepted date: August 18, 2016; Published date: August 28, 2016

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The anaerobic fermentation required for ethanol production from syngas appears to be a promising and competitive engineering application technology. The main challenge facing for syngas fermentation is the fact that it is often limited by low ethanol productivity. To evaluate the ability of different syngases and microorganisms to ferment ethanol and screen those efficient syngases and microorganisms, three simulative syngas mixtures and eight strains were investigated in 300 ml bottle fermentations. The results show that, both syngases and cultures affected ethanol production at a highly significant level (p<0.01). The maximum net ethanol concentrations (28.001, 23.871, 22.909 and 19.726 mg/L) were obtained with strains LP-fm4, C. carboxidivorans P7, B-fm4 and C. ljungdahlii using biomass-generated syngas, which yielded approximately two and three times more ethanol compared with strains C. ljungdahlii, B-fm4 (11.734, 10.300 mg/L) using corex-gas and strains C. carboxidivorans P7, C. ragsdalei P11 (9.937, 8.318 mg/L) using blast furnace gas, respectively. So, using biomass-generated syngas and strains LP-fm4、C. carboxidivorans P7、B-fm4 and C. ljungdahlii represented the best combination for fermenting ethanol efficiently compared with the other two. In addition, the maximum ethanol production per unit cell of both strains LP-fm4 and B-fm4 with biomass-generated syngas, C. ragsdalei P11 with blast furnace gas and B-fm4 with corex-gas were 1000.036, 881.103, 519.854 and 468.030 mg/L, respectively. This indicates that strains LP-fm4 and B-fm4 are the most promising for biomass-generated syngas fermentation, strains C. ragsdalei P11 and B-fm4 are potential candidates for blast furnace gas and corex-gas fermentations respectively.


With the rapid growth in the world’s population and the increasing development of industrialization, more and more energy resources are required eagerly. According to calculated data, the population of our planet for the year 2050 is estimated to be in excess of 9 billion [1]. In addition, fossil fuels containing oil and natural gas, are being rapidly depleted, total reverses to the end of year 2012 are approximately able to support the energy consumption for the next 51 and 56 years respectively [2]. On the other hand, the burning of fossil fuels has caused an increase in greenhouse gas emissions that causes global warming, acid-rain and urban smog, etc. So these negative environmental consequences of fossil fuel consumption and concerns about petroleum supplies have spurred the search for new, more sustainable and renewable energy sources [3-6]. Ethanol is one of the most promising alternative biofuels, which provides a net energy gain, has environmental benefits and is economically competitive. For instance, the US biofuels industry is undergoing rapid growth and transformation, it has mandated the production of 36 billion gallons of biofuels by 2022 [7,8] while China will produce 500 million tons of ethanol based by 2015. Planned production of ethanol will expand to 1 billion tones by year 2020. However, this bioethanol is mostly derived from food sources of feedstock such as sugar, corn or starch, which may become a hidden threat to global food security in the future.

Luckily, fermentation of syngas or waste gas components to produce ethanol appears to be a promising alternative compared to existing chemical techniques [9-11]. Syngas is a mixture of principally CO, CO2 and H2, which can be produced by gasification of solid fuels(such as coal, oil shale, petroleum coke, biomass or organic wastes from daily life, etc)[9,12-16]. Moreover, with the growth in the iron and steel industries, more and more exhaust gases are also used as syngas through pre-treatment processes: these are directly emitted into the atmosphere generally. Compared to other conversion technologies, syngas fermentation offers several advantages[10,17]. However, the phenomenon facing current syngas fermentation is low ethanol productivity caused by low gas-liquid mass transfer, syngas composition and the fermentation microorganisms themselves.Of these three factors, the former has been studied more[18-21], and the latter two are relatively less . To date, steel waste gases such as blast furnace gas and corex gas have not been studied further as the amount of fermentation strains found is too small. Bacteria used in the hybrid conversion process are called acetogens, which are anaerobes that assimilate syngas fermenting ethanol via the Wood-Ljungdahl pathway. Examples of cultures used are Clostridium ljungdahlii[22,23], Clostridium carboxidivoransP7[24,25], Clostridium ragsdalei P11[26], Clostridium autoethanogenums[27], Alkalibaculumbacchi strains CP11T, CP13[28], etc. Meanwhile, this research groups has isolated and enriched four dominant microflorae from animal faeces samples of alpaca, gibbon, lesser panda and papion with biomass syngas under strict anoxic conditions, these are A-fm4, G-fm4, LP-fm4, B-fm4 respectively.

To evaluate the ability of different syngases and microorganisms to ferment ethanol and screen the efficient syngases and microorganisms improving ethanol production in syngas fermentation, this study was designed to investigate three simulative syngas mixtures (biomass-generated syngas, blast furnace gas and corex gas) and eight strains (A-fm4, G-fm4, LP-fm4, B-fm4, Clostridium autoethanogenumsDSM10061, C.ljungdahlii, Clostridium carboxidivoransP7 and Clostridium ragsdaleiP11) in 300ml bottle fermentations.

Materials And Methods

Microorganisms, Syngases and Media

The bacteria used in this study were A-fm4, B-fm4, G-fm4, LP-fm4, Clostridium autoethanogenumsDSM10061, C.ljungdahlii(ATCC 55383), Clostridium carboxidivoransP7 and Clostridium ragsdaleiP11 respectively. A-fm4, B-fm4, G-fm4 and LP-fm4 were isolated in a laboratory. The genomic DNA of the four samples was extracted and sequenced, from which their phylogenetic tree was constructed. Analysis shows that the main microorganism in the four dominant microflora are anaerobic bacterium, bacillus and coccus in A-fm4, bacillus and enterobacter in G-fm4 and B-fm4, and bacillus in LP-fm4. Like the Clostridium autoethanogenums, the four dominant micro flora are gram-positive, and the main bacterium contained in the dominant microflora are rod-like. G-fm4 is mostly like DSM10061; both are rod-like, gram-positive and spore-forming. Moreover, C. autoethanogenumsDSM10061 was acquired from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), cultures C.ljungdahlii(ATCC 55383);Clostridium carboxidivoransP7 and Clostridium ragsdaleiP11 were provided by The Key Laboratory of Synthetic Biology (Chinese Academy of Sciences, Shanghai, China). These strains were maintained on their respective growth media as reported previously[14], and allowed to grow under strict anoxic conditions at 37°C,at a pH of 5.75.

Three artificial syngas mixtures obtained from Yuan Zheng Development Company (Zhengzhou, China) were investigated in this study. The first syngas, a biomass-generated syngas, contained 85.5% CO, 10% H2 and 4.5% CO2 by volume; its composition simulates the gas from maize straw after gasification. The latter two syngases(blast furnace gas and corex gas), are by-products of the iron-steel industry, and simulate a gases from Baosteel (Baosteel Group Corporation, Shanghai, China). Blast furnace gas contained 22.4% CO, 23.6% CO2, 3.3% H2, 50.6% N2 and 0.1% O2 by volume. Corex gas containe 17.72% H2, 45.23% CO, 33.17% CO2, 1.68% CH4, 2.20% N2 and 100 to 120ppm H2S.

The fermentation medium[29] used contained (per litre) :10ml mineral solution[14], 10ml vitamin solution[14], 1.0g NH4Cl, 1.0g NaCl, 0.15 g MgSO4, 0.1 g KH2PO4, 0.04 g CaCl2, 2.0 g Tryptone, 0.3g yeast extract, 10.0g 2-(N-morpholino)ethanesul-fonic acid (MES), 0.1ml 0.1% resazurin solution, and 0.2g/L cysteine-HCl for use as a reducing agent. The pH of the medium was adjusted to 4.5 using 10M NaOH. Vitamin solution and cysteine sterilized by filtration, and then added to the medium after autoclaving. The medium was firstly prepared by boiling for a few minutes, while being degassed, and then cooled continuously in an YX-IIanaerobic box (Shanghai Medical Equipment Manufacturing Co., Ltd, Shanghai, China) for 24 hours to remove any oxygen. Finally, the media in bottles were sterilized at 121°C for 20 min. All experiments were carried out in triplicate.

Fermentation Runs

Batch fermentations were done in 300 ml serum bottles each containing 60 ml of fermentation medium. 10% (v/v) of inoculum was transferred to fresh media. The cultures were maintained under anaerobic conditions and agitated at 150 rpm on an QHZ-98A orbital shaker (Huamei Biochemical Instrument Company, Taicang City, China ), inside an incubation chamber at 37°C, which were grown with three simulative syngas mixtures as the sole substrate. The syngas inlet volume was 240 ml, injected in each 300 ml serum bottle by syringe. Experiments were conducted every 24 h for 5 days, samples (2.0 ml) were collected to analyse pH and growth. Another 1.5 ml sample was centrifuged (10000 rpm, 10 min) to remove the cells; the supernatant was used to measure ethanol production.

Statistical Analysis

Analysis of variance (ANOVA) was undertaken using SPSS 16.0 software for ethanol production based on three simulative syngas mixtures and eight strains, it was used to determine if statistical significant differences exist in ethanol production, organisms, syngases, the maximum ethanol production and ethanol production per unit cell between the treatments with the strains and syngas mixtures used at the 95% confidence level. In addition, a t-test was also employed to determine whether or not values were significantly different for the maximum ethanol production and ethanol production per unit cell compared with the control strain C. autoethanogenums DSM10061. Ethanol production per unit cell [30] was calculated as follows:

Ethanol production per unit cell = Equation

Where the maximum ethanol production*, Initial ethanol production* and Maximum cell mass (OD600)* are the mean of three observations, respectively.

Analytical Procedures

Growth was monitored by measuring optical density (OD) at 600 nm with a 722S spectrophotometer (Qinghua Scientific Instruments Co., Ltd., Shanghai, China). Ethanol concentration was analyzed using Agilent 7890A GC system (Agilent Technologies, Wilmington, DE, USA) with a flame ionization detector (FID) and an HP-FFAP capillary column (30 m × 0.32 mm × 0.3 μm) (Agilent Technologies, Wilmington, DE, USA). The sample size was 0.2 μL. Nitrogen was used as carrier gas at flow rate of 1.5 mL/min. The inlet port temperature was kept at 200°C with a split ratio 30:1. The initial oven temperature was set at 45°C with a holding time of 1.0 min. It was then increased at a ramping rate of 10°C/min to 80°C with a holding time of 0.5 min. The FID temperature was set to 250°C with hydrogen and air flow rates of 30 ml/min and 350 ml/min, respectively.


Cell Growth and Effect

The growth profiles of eight cultures using three syngas mixtures in batch fermentations are shown in Figure 1. It is observed that eight strains nearly had a decreasing trend during fermentation. Figure 1A shows there is a minor change in cell mass concentrations of the strains except for Clostridium carboxidivoransP7, which started to grow until one day had elapsed and attained a maximum concentration (OD600=0.061), then decreased rapidly until the fourth day. However, compared to biomass-generated syngas fermentation, eight organisms had a similar growth trend as shown in Figures 1B and 1C. Additionally, strains G-fm4, C. autoethanogenums DSM10061 and Clostridium ragsdalei P11 started to grow until the second,third and fourth days respectively (Figure 1B). For cultures A-fm4, B-fm4 and LP-fm4, they all reached their maximum cell level at the end of the second day, while control strain C. autoethanogenums DSM10061 reached the same piont after the fourth day (Figure 1C).

Soil pH(H2O) Total N Total C CECa Available P Available K
    ------- g kg-1 -------   cmol+ kg-1 ------------ mg kg-1 ------------  
C 6.71 2.4 22.2 27.5 17.1 1964
F 5.54 3.1 26.3 5.3 66.7 137

Table 1: Chemical properties of the investigated topsoil samples (< 2 mm).


Figure 1: Growth of organisms in batch culture with syngases. A, eight cultures with biomass-generated syngas. B, eight cultures with blast furnace gas. C, eight cultures with corex gas. Error bars (n=3) represent plus and minus one standard deviation from the average of every experimental replicates.

Product Formation and Effect

Figure 2A shows the ethanol production profiles of eight strains using biomass-generated syngas in batch cultures. The maximum ethanol concentration (82.006mg/L) was produced by Clostridium ljungdahliiat the end of the third day; this concentration was significantly higher than in the other cases. The cultures, except Clostridium ljungdahliicould all attain their peak of ethanol concentration by the end of the fourth day. For organisms, Clostridium carboxidivoransP7, LP-fm4 and B-fm4, the ethanol concentration in batch fermentation were 56.386, 51.729 and 46.922 mg/L, respectively, which also increased significantly compared with strains A-fm4, G-fm4, Clostridium ragsdalei P11 and the control. Additionally, only strains A-fm4, B-fm4, LP-fm4, Clostridium carboxidivoransP7 and Clostridium ragsdalei P11 were observed to produce ethanol during fermentation in the eight cultures (Figure 2B). The strain Clostridium carboxidivoransP7 had the highest ethanol concentration at 38.282 mg/L in a manner similar to Figure 2A. Figure 2C shows that the ethanol concentration peaked at the end of the fourth day (53.866 mg/L) and subsequently decreased on the fifth day for all strains, it can be seen that the cultures producing ethanol mainly occurred as mid to late-batch fermentations. Statistical analysis indicated that ethanol production fermented by eight cultures using each kind of the three artificial syngas mixtures had a high significant effect (P< 0.01) ,as shown in Tables 1 to 3.

Treatments Soil Antibiotics Concentration (mg kg-1)
C C none 0
CP10 C Penicillin G 10
CP100 C Penicillin G 100
CT10 C Tetracycline hydrochloride 10
CT100 C Tetracycline hydrochloride 100
F F none 0
FP10 F Penicillin G 10
FP100 F Penicillin G 100
FT10 F Tetracycline hydrochloride 10
FT100 F Tetracycline hydrochloride 100

Table 2: Antibiotics concentration (mgkg-1) in treated soils.

Treatment 1d 3d 7d 14d 27d
    Bacteria (107cfu g-1 dry soil)  
C 73.5a 64.0a 65.2a 40.7a 44.0a
CP10 55.0b 48.0b 49.5b 40.5a 45.7a
CP100 33.5c 34.2c 38.5c 33.2b 42.2a
CT10 45.0b 43.7b 49.0b 23.7c 47.2a
CT100 34.7c 37.5c 41.5bc 32.0b 42.0a
F 72.5a 64.5a 55.7a 46.7a 48.5bc
FP10 41.2b 33.2b 29.0b 28.2b 60.0a
FP100 26.0c 23.7c 14.7c 25.5b 55.0ab
FT10 43.2b 39.7b 29.7b 26.7b 54.5ab
FT100 39.5b 39.2b 14.5c 20.0b 40.7c
    Fungi (104 cfu g-1 dry soil)  
C 40.0a 55.0a 47.5a 45.0a 42.5b
CP10 30.0b 45.0ab 37.5b 42.5ab 55.0a
CP100 17.5c 25.0c 40.0ab 35.0b 45.0ab
CT10 37.5a 40.0b 35.0bc 27.5c 35.0c
CT100 22.5c 20.0c 30.0c 42.5ab 45.0ab
F 35.0a 52.5a 45.0a 32.5a 20.0b
FP10 27.5b 27.5b 22.50b 25.0b 20.0b
FP100 15.0cd 9.0d 7.5b 7.5c 27.5a
FT10 20.0bc 27.5b 20.5b 27.5ab 22.5b
FT100 12.5d 20.0c 15.5b 20.0b 32.5a
    Actinomycetes(104 cfu g-1 dry soil)  
C 46.0a 40.2ab 39.0b 35.5a 45.7a
CP10 45.2a 37.0abc 44.5ab 34.5a 30.7b
CP100 50.7a 43.2a 37.7b 30.5a 23.5 c
CT10 56.5a 36.0bc 51.5a 38.0a 39.7b
CT100 51.7a 34.0c 37.0b 35.7a 35.7b
F 45.50a 40.5b 34.0a 33.2a 25.2a
FP10 35.2b 43.7ab 30.5a 32.0a 9.7c
FP100 43.2ab 52.2a 33.0a 33.0a 16.2b
FT10 24.0b 43.5ab 35.7a 26.2a 15.2b
FT100 44.7a 43.7ab 34.5a 27.2a 15.5b

Table 3: Populations of bacteria, fungi and actinomycetes in soil after penicillin and tetracycline treatments.


Figure 2: Ethanol production. A, eight cultures with biomass-generated syngas. B, eight cultures with blast furnace gas. C, eight cultures with corex gas. Error bars (n=3) represent plus and minus one standard deviation from the average of every experimental replicates.

Ethanol Production Capacity

Figure 3 compares ethanol production capacity between seven organisms and the control strain Clostridium autoethanogenumsDSM10061 using three artificial syngas mixtures. Ethanol production is a process of accumulation during fermentations and the ethanol production per unit cell introduced could be used as a measure of the fermentative capability of unicellular ethanol production. In Figure3A, significant differences (P<0.01) were observed in maximum ethanol production with strains B-fm4, LP-fm4, Clostridium carboxidivoransP7, Clostridium ljungdahliiand Clostridium autoethanogenumsDSM10061. However, the ethanol production per unit cell with the control strain and Clostridium ljungdahliiwas significantly lower than with B-fm4 and LP-fm4. Similarly, there were significant differences (P<0.01) in maximum ethanol production with strains B-fm4, G-fm4 LP-fm4, Clostridium carboxidivoransP7 and Clostridium ljungdahliicompared to the control while the ethanol production per unit cell with culture B-fm4 had become the highest(Figure 3C). Conversely, Figure 3B shows that only six strains could ferment ethanol, in which cultures Clostridium carboxidivoransP7 and Clostridium ragsdalei P11 had a higher ethanol production capacity


Figure 3: Comparison of the maximum ethanol production and ethanol production per unit cell for strains with syngases. A, eight cultures with biomass-generated syngas. B, eight cultures with blast furnace gas. C, eight cultures with corex gas. Error bars (n=3) represent plus and minus one standard deviation from the average of every experimental replicates.

Moreover, to compare ethanol production capacity of organisms with syngases in the present study distinctly, Table 4 lists the net ethanol production fermented by syngases and cultures during fermentations. The maximum net ethanol concentrations (28.001, 23.871, 22.909 and 19.726mg/L) were obtained with biomass-generated syngas and strains LP-fm4,C. carboxidivoransP7,B-fm4 and C.ljungdahlii, which was about twofold and threefold more ethanol compared with corex-gas and strains C.ljungdahlii, B-fm4 (11.734, 10.300mg/L) and blast furnace gas and strains C. carboxidivoransP7 and C. ragsdaleiP11 (9.937, 8.318mg/L) respectively. However, the net ethanol levels of strains G-fm4, Clostridium ljungdahliiand Clostridium autoethanogenumsDSM10061 were all zero. Furthermore, the difference in the effect of amounts of ethanol formed by the different organisms and syngases was highly significant(P<0.01), as shown in Table 5, which indicated that both played a major role in ethanol fermentations.

Syngasesa The averagevalue of net ethanol production during fermentation (mg /L)
Organismsb 1 2 3 4 5 6 7 8
BMG   14.018 22.909 10.411 28.001 23.871 10.548 19.726 1.833
BFG   0.632 0.189 0 0.184 9.937 8.318 0 0
CG   3.429 10.300 2.657 3.741 3.710 4.462 11.734 3.181

Table 4: The net ethanol production fermented by syngases and organisms during culture.

Source Sum of squares df Mean square F F0.01
Syngases 2611.894 2 1305.947 61.443** 4.98
Organisms 904.728 7 129.247 6.081** 2.95
Error 1317.757 62 21.253    
Total 4834.379 71      

Table 5: ANOVA table: net ethanol production obtained by syngases and organisms


    In this research, we firstly have evaluated the ability of different syngases and microorganisms to ferment ethanol and screened the efficient syngases and cultures using three simulative syngas mixtures and eight strains. Both played a major role in ethanol fermentations based on statistical analysis, which affected ethanol production highly significant (p<0.01). As shown by the data, it was found that the maximum net ethanol concentrations (28.001, 23.871, 22.909 and 19.726mg/L, respectively) were obtained by strains LP-fm4、C. carboxidivoransP7、B-fm4 and C.ljungdahliiwere generated when using biomass-generated syngas. Compared with strains C.ljungdahlii, B-fm4 using corex-gas and cultures C. carboxidivoransP7, C. ragsdaleiP11 using blast furnace gas, the combination of using biomass-generated syngas and strains LP-fm4、,B-fm4 and C.ljungdahliiwill be optimal for efficient ethanol fermentation. As reported in results arising from this research, cultures LP-fm4、C. carboxidivoransP7、B-fm4 and C.ljungdahliiwere known as ideal bacterias for the use of biomass syngas in ethanol fermentation. Additionally, the maximum ethanol production per unit cell of strains LP-fm4 and B-fm4 using biomass-generated syngas were 1000.036, and 881.103 mg/L respectively (Figure 3A) while the cell concentrations of both organisms were lower than the other two strains, shown as Figure 2A . Strains LP-fm4 and B-fm4 with increasing cell concentrations in ethanol fermentations will in some way produce higher ethanol concentrations using biomass syngas in the future. So, these data laid the foundation for biomass-generated syngas fermentation, further research is required and this may have a great significance in promoting comprehensive utilisation of biomass resources and propelling ethanol fermentations with biomass syngas the forward.

In contrast, ethanol productions as fermented by strains using syngas mixtures with blast furnace gas and corex-gas were lower than with biomass syngas (Figure 2 and Table 4), this may have had a close relationship to the components in syngases. For instance, Liu and Atiyeh studied ethanol production from two commercial syngas mixtures (Syngas I: 20% CO: 15% CO2: 5% H2 and 60% N2: SyngasII : 40% CO: 30% CO2 and 30% H2) using three moderately alkaliphilic strains, results showed that strains produced more ethanol with syngasII than syngasI [28]. In this research, three syngas mixtures were used, which were biomassgenerated syngas, blast furnace gas and corex gas. Their components varied, biomass syngas contained 85.5% CO, 10% H2, 4.5% CO2, and blast furnace gas contained 22.4% CO, 23.6% CO2, 3.3% H2, 50.6% N2, 0.1% O2 and corex gas contained 17.72% H2, 45.23% CO, 33.17% CO2, 1.68% CH4, 2.20% N2 and 100 to 120 ppm H2S. These observations indicated that carbon monoxide was a major component in synthesis gas for fermenting ethanol efficiently. For example, it was seen that the ethanol productions of strain B-fm4 gradually increased using biomass syngas, blast furnace gas and corex gas, respectively, the concentrations of carbon monoxide in the syngases also increased (Figure 3). Similarly, as reported in the literature [12,26,28,31,32], the authors found that the maximum cell concentration and ethanol production were associated with increasing PCO. So, it was thought here that the high proportion of CO in syngas mixtures would benefit ethanol fermentations. Theoretically, all the carbon in CO is converted to ethanol at an H2:CO ratio of 2 (Eq.(2) and (3)); but, only a third of the carbon in the CO is converted to ethanol without H2 (Eq.(1)). Here, H2 served as an electron source, which can be converted to reducing power by hydrogenase when H2 is present.

6CO+3H2O→C2H2OH+4CO2 △G0= -217.8 kJ/mol   (1)

2CO+4H2→C2H5OH+ H2O △G0= -137.6 kJ/mol   (2)

2CO+6H2→C2H5OH+ 3H2O △G0= -97.5 kJ/mol   (3)

More carbon from CO will be converted to ethanol instead of CO2 at this time. Conversely, never was an increase in both ethanol production and growth with higher hydrogen rate (H2-rich condition) observed, which was different from the theoretical prediction made before carrying out the experiments. It was considered that the major causes leaded to the situation above contained two aspects, one was the presence of carbon monoxide in syngas mixtures, what was a known inhibitor of hydrogenase [31,33,34], and could inhibit the utilisation of hydrogen by the organism. Another was the pH during fermentations. The pH used in this work was 4.5, which was considered relatively good for ethanol production [29,35]. However, other research [36]showed that the optimum pH for the in vitro measurements of hydrogenase activity was found at pH 8.5, and there was inactivity at pH values below 6.0 to be detected. So, the hydrogenase in the solvent-producing cells (grown at pH 4.5) was presumably present in an inactive form druing this research. In addition, the results presented in Figure 3B and Table 4 demonstrated that strains G-fm4, C.ljungdahlii and the control could not produce ethanol using blast furnace gas containing 0.1% O2 by volume. This indicated that the three cultures were all too sensitive to oxygen to live while strains A-fm4, B-fm4, LP- fm4, C. carboxidivorans P7 and C. ragsdalei P11 had some O2 tolerance, which may be the first published evidence to this effect to date. Moreover, compared to organisms using biomass syngas, the ethanol production of strains using blast furnace gas was lower, which also suggested that the oxygen present in the syngas could inhibit growth and ethanol formation in fermentations especially as the concentration of O2 was above the tolerable maximum.

    For blast furnace gas and corex gas, low calorific value residual gases generated in iron and steel industries [37-39], these by-product gases are traditionally emitted into atomosphere, this causes air pollution. From the finding that the maximum net ethanol concentrations of strains C. carboxidivorans P7 and C. ragsdalei P11 using blast furnace gas and cultures C.ljungdahliiand B-fm4 using corex gas were 9.937, 8.318mg/L and 11.734, 10.300 mg/L, respectively, it deduced that the strains above could grow and produce ethanol based on these syngases. In addition, the maximum ethanol production per unit cell of strains C. ragsdaleiP11 and B-fm4 (Figures 3B and 3C) indicated that both were considered as the ideal potential candidates for blast furnace gas and corex-gas fermentations respectively. To produce commercial quantities of ethanol, it may be possible to optimise the composition of the medium to obtain higher concentration strains, or deploy gas compositions based on the practical production needs. Otherwise, it is known that the temperature of these exhaust gases is relatively high, it would be interesting and meaningful to find a suitable thermo-phile for ethanol production from these waste gases. Furthermore, the amount of carbon dioxide in these waste gases will be increasing with the technological development of iron and steel industries, and CO2 was also one of the main components in syngas. Hence, syngas fermentation will be applied to blast furnace gas and corex-gas more efficiently and become a promising and competitive method of reducing environmental pollution and promote the comprehensive utilization of industrial waste gas resources.

The syngases used in this work were all simulated mixtures, which only contained the key components of an actual syngas whose actual components were diverse and complex. For instance, biomass syngas is generated by the gasification of biomasses, and is a mixture of CO, H2, CO2, N2, CH4, NOX, O2, CH4,C2H2,C2H4,H2S, NH3 and tars, etc[9,31,40]. Besides, both blast furnace gas and corex-gas also contain: H2, CO2, N2, H2S, NOX, O2, etc [41]. However, these impurities can potentially affect the fermentation process. In this work, it was shown that accumulated O2 in the mixtures could inhibit cell growth and ethanol production (Figures 1B and 3B) and as and impurity, H2S also reduced the ethanol production (Figure 3C). At the same time, Ahmed found that tars promoted cell dormancy of strain C. carboxidivoransP7 and a redistribution of ethanol and acetic acid production[42], a similar result to that found by Guo[43] using strain Clostridium autoethanogenumsDSM10061. Ahmed also studied the effect of NO on the fermentation of biomass syngas with strain C. carboxidivoransP7, the result showed that nitric oxide could inhibit hydrogenase and preventsutilisation of H2 by this strain when the concentration of NO exceeded 40ppm, it therefore acted as a non-competitive inhibitor of hydrogenase activity[44]. Ammonia (NH3) is another component in syngas; it can rapidly convert to NH4+ following exposure of fermentation media to NH3, which transports across the cell membrane to inhibit hydrogenase. A recent report[40]indicated that NH4+ is also a non-competitive inhibitor for hydrogenase activity with K NH4+ of (649±35) mol m-3. Besides, methane (CH4) is also present in the corex gas used in this work at a centration of 1.68%(v/v). However, the effect of CH4 on cell growth and ethanol production has not been observed, nor was the utilisation of CH4 has been detected here. Only a study using C. ragsdaleiP11 showed that CH4 was not consumed at contents up to 5%(v/v), which did not affect cell growth or ethanol production[31]. So, syngas components were complex and the effects of these impurities in syngas were not to be neglected, but whether or not other impurities have the similar effects on fermentation strains remains to be proven. Meanwhile, further research should quantify these impurities in syngases and assess their impact on the current experimental results.


Studies were conducted to evaluate the ability of different syngases and microorganisms to ferment ethanol, the most efficient syngases and cultures using three simulative syngas mixtures and eight strains in batch fermentations were selected. Findings from this study indicated that both syngases and cultures affected ethanol production to a significant extent (p<0.01). The ethanol production fermented by strains LP-fm4,C. carboxidivoransP7,B-fm4 and C.ljungdahliiusing biomass-generated methods was higher than the two other methods, strains C. carboxidivorans P7 and C. ragsdalei P11 were fit for blast furnace gas fermentation as well as organisms C.ljungdahlii,and B-fm4 , which were suited to corex gas fermentation under the experimental conditions described here. Compared with three simulated syngas mixtures used here, the high concentration of CO in syngas would benefit ethanol fermentations. Additionally, strains LP-fm4 and B-fm4 were the most promising for biomass-generated syngas fermentation; culturessC. ragsdalei P11 and B-fm4 are ideal potential candidates for blast furnace gas and corex-gas fermentations respectively according to the value of maximum ethanol production per unit cell. It is expected that the choice of strains fermenting syngas to ethanol production and the deployment of syngas components will further enhance ethanol yield for potential commercial use.


This research was financially supported by the program for New Century Excellent Talents in The University of The Ministry of Education of China (Grant no. NCET-12-0695) and the program for Innovative Research Team (in Science and Technology) in University of Henan Province (No.15IRTSTHN014).