2014 Volume 20 Issue 4 Pages 867-873
Bacterial diversity of the aged and aging pit mud from Luzhou-flavor liquor distillery was investigated and compared by molecular methods. Two bacteria-specific 16S rRNA gene clone libraries were constructed and analyzed using the amplified rRNA gene restriction analysis. A total of 172 clones were studied and 23 operational taxonomic units (OTUs) were obtained. Firmicutes and Chloroflexi predominated in the aged pit mud while Firmicutes and Bacteroidetes predominated in the aging pit mud. What is more, Chloroflexi and Actinobacteria were only detected in the aged pit mud. Additionally, the results of quantitative real time PCR (qPCR) showed that the quantity of Actinobacteria in the aged pit mud was 29 times as much as in the aging pit mud (P < 0.05). The results have compared the bacterial diversity of the aged and aging pit mud for the first time and maybe useful to further understand the microorganism of pit mud.
Chinese liquor is one of the six famous distilled alcoholic beverages including Brandy, Whisky, Rum, Vodka, Gin and Spirit in the world and their producing regions are France, England, Cuba, Russia, Holland and China, respectively (Sheng et al., 2007). Luzhou-flavor liquor is a typical representative of the Chinese intense fragrance liquors (Xiang et al., 2013). Many famous strong aromatic liquors are all fermented from grains in a soil cellar (pit) which is a rectangular underground pool constructed by pit mud. The pit mud (i.e. specific fermented clay) provides a suitable habitat for the brewing microbiota (Zheng et al., 2013). Hence, the pit mud of cellars plays an important role in the production of Luzhou-flavor liquor (Zheng et al., 2012).
Recently, the microbial study of pit mud mainly concentrated in many famous liquor distilleries, for example, Luzhoulaojiao (Deng et al., 2012; Ding et al., 2013). In china, the Luzhou-flavor liquor distilleries distribute extensively, so there are some differences in the micro-environment of pit mud. Therefore, the investigation of pit mud in different areas is useful to reveal the characters of pit mud and systematically understand the microbial diversity of pits.
According to the traits, pit mud can be divided into the aged and aging pit mud. The aged pit mud is moist, ebony, soft and crisp with a rich aroma and a clear smell of hydrogen sulfide and ammonia odor (Zhang et al., 2012). It could produce high quality liquor. In the fermenting process of Luzhou-flavor liquor, the aged pit mud gradually hardens and white crystals or elongated needle crystals appear on its surface after a period of time. At this time, the aging pit mud is formed. It is light gray, hard, stingy and dry and contains white lumps or crystals and lacks flavor (Jiang et al., 2008). In addition, the aging pit mud is unfavourable to the flavor and taste of final liquor products. But the recent microbial studies on pit were focused on pit mud of cellars used for different periods of time, litter is known about the difference between the aged and aging pit mud. Therefore, it is necessary to explore the difference of microbial community structure between the aged and aging pit mud for improving the quality of Luzhou-flavor liquor.
Previous studies concerning microbial community structure have been performed based on culture-dependent methods (Jiang et al., 2008). However, most of the microorganisms are uncultured or difficult to culture, and culture-dependent method is difficult to reveal the inner pattern comprehensively and objectively (Amann et al., 1995). Recently, molecular ecological methods, such as denaturing gradient gel electrophoresis (DGGE), 16S rRNA libraries, amplified rRNA gene restriction analysis and quantitative real time PCR (qPCR) (Bowers et al., 2000) are widely used to analyze microbial community structures in complex environments. For instance, Deng et al. analyzed microbial community evolution in the pit mud of cellars used for different periods of time by PCR-DGGE (Deng et al., 2012; Shi et al., 2011). Although there are many advantages in using PCR-DGGE for microbial community analyses, method limitations should be recognized for correct result interpretations. For example, the limitation of DGGE is that a complex community (e.g. soil) may be comprised of numerous populations (from > 100 and possibly > 108) in relatively equivalent proportions, thus resulting in a smear of bands, which makes it difficult to identify individual populations (Nakatsu et al., 2007). Nevertheless, DNA libraries for the microbial 16S rRNA can cover the total microbial diversity or the diversity within distinct phylogenetic groups (Klocke et al., 2008). Moreover, the analysis of 16S rRNA libraries is commonly combined with amplified rRNA gene restriction analysis, which could reduce sequencing efforts and was regarded as sufficient for resolving even small differences between microbial species (Klocke et al., 2008). Meanwhile, the recombinant plasmids obtained from 16S rRNA libraries could be used for standard curve analyses of qPCR. Furthermore, qPCR could be applied to the accurate determination of the abundance of certain 16S rRNA groups within a pool of microbial DNA (Klocke et al. 2008). The qPCR approach could be used for the additional determination of the accurate quantity of detected taxonomic units.
To date, little is known about the difference of microbiological information in the aged and aging pit mud. Therefore, in the present study, the bacterial structure between the aged and aging pit mud from Anhui Province has been investigated by 16S rRNA gene libraries, amplified rRNA gene restriction analysis and qPCR.
Sample collection Samples of the pit mud were collected at the bottom of the pit from a major liquor-making factory affiliated to the Golden Seed in Anhui Province, China. The pit mud, about 2000 grams, were taken from the four corners and the center of the bottom of the pit and then mixed. According to the traits, pit mud samples were distinguished into aged and aging pit mud. The samples were transferred with an ice-cooled sample taker to sterile polyethylene bags, which were sealed and stored at −20°C until used.
DNA extraction and PCR amplification of 16S rRNA The total DNA was extracted from the samples by soil DNA kit (Omega, USA) according to the manufacturer's instructions. PCR was carried out in a Mastercycler (Bio Rad, USA). Each reaction mixture contained 2 × PCR Mix (Sangon Biotech, Shanghai, China), oligonucleotide primer (20 µmol/L) and distilled water to a final volume of 50 µL.
An approximate 1500 bp fragment of bacterial 16S rRNA from the preparations of the total microbial DNA was amplified by PCR using forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3') (Ribeiro et al., 2013). Approximate 10 ng of three pooled DNA preparations was used as templates. Reaction mixtures were denatured at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, with a final extension step of 10 min at 72°C. The PCR product was analyzed by gel electrophoresis and purified for subsequent cloning with a commercial kit (Sangon Biotech, Shanghai, China).
Construction of 16S rRNA libraries and amplified rRNA restriction analysis The 16S rRNA amplicons of bacteria were cloned into pUCm-T plasmids (Sangon Biotech, Shanghai, China) and then transformed in E.coli DH5α competent cells (Tiangen, Beijing, China) according to the manufacturer's guidelines (Sangon Biotech, Shanghai, China). For the sake of reducing the bias caused by PCR, the products of three single PCR reactions were pooled, as previously recommended by several authors (Sekiguchi et al., 1998). Clones were plated on Luria-Bertani agar medium containing with 100 µg/mL ampicillin (Sangon Biotech, Shanghai, China), 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (Amresco, USA) which Cat# was 0428 and Isopropyl β-D-1-Thiogalactopyranoside (Sangon Biotech, Shanghai, China) which Cat# was IB0168. After one night at 37°C, white clones were picked for positive tests. Each white clone was inoculated in the PCR buffer (containing 2 × PCR Mix, primers as described before) using sterile toothpick. Resulting PCR products were checked for size and quality on 2% agarose gels (Biowest, Spain) containing ethidium bromide and molecular ladder (Sangon Biotech, Shanghai, China).
PCR products of positive recombinant plasmids were digested with Hha I enzyme (Fermentas, China) for 2 h at 37°C. This enzyme has proven to be efficient to describe microbial diversity in previous studies (Cambon-Bonavita et al., 2001). Reaction conditions were: 100 to 500 ng of DNA, 1 × final buffer concentration as recommended and 5 U of the enzyme. Restriction results were examined on 2% agarose gels containing ethidium bromide and 100-bp molecular ladder as mentioned above. Pictures of gels were made with the Eletrophoresis (Biorad, Beijing, China).
Sequencing and sequence comparisons One of each different profile obtained from Hha I restriction was selected for further studies. Clones were cultivated in 1 mL Luria-Bertani liquid medium with ampicillin (100 µg/mL) liquid medium at 37°C for one night. Plasmid extraction was carried out using the Mini Plasmid Kit (Tiangen, Beijing). Each plasmid DNA was sent to the Sinogenomax Company Limited for sequencing. Individual amplified patterns were used as OTU. Chimera Check software was used to exclude chimeras. The 16S rRNA sequences obtained were compared with those in Gen Bank Database using the BLAST (i) program and the percent similarity was then determined. Multiple alignments were carried out using the Clustal X program (Thompson et al., 1997). The Phylogenetic trees were constructed with the MEGA program version 5.0 using the neighbor-joining method with the Kimura two-parameter model (Tamura et al., 2011). All bacterial 16S rRNA sequences from the OTUs were respectively classified by program RDP (ii) Seqmatch and GeneBank Database. The coverage of clone libraries which could be used to reflect whether clone numbers was satisfactory or not, was determined using the formula (as Eq. 1), with n as the number of phylotypes (OTUs) represented by one single clone and N as the total number of clones (Leser et al., 2002).
In order to estimate the diversity of bacteria, the Shannon index (H) was calculated as Eq. 2, where ni is the number of individuals of taxon i, and n the total number of organisms of all species (Shannon, 2001). The index gives the proportional abundance of species and reacts sensitively to rare species. When all species are represented by the same number of individuals, the index reaches its maximum value. In order to describe the uniformity of the distribution of the individuals on the number of OTUs, the evenness was calculated (as Eq. 3). The Shannon diversity index and evenness were calculated with the software BIO-DAP. Rarefaction analysis, displaying the number of OTUs detected versus the number of clones analyzed, was performed using the Sigma Plot Software.
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Actinobacteria 16S rRNA gene copies by qPCR According to the results of 16S rRNA libraries, Actinobacteria was only detected in the aged pit mud. Therefore, the quantity of Actinobacteria might be the important mark of the aged pit mud. Actinobacteria was quantified by the qPCR amplification of the 16S rRNA gene. qPCR was performed using SYBR Green I and conducted in a Lightcycler Nano System (Rocha, Switzerland). Primers for the genera Actinobacteria were Act920F (5'-TACGGCCGCAAGGCTA-3') and Act1200R (5'-TCRTCCCCACCTTCCTCCG-3') (Bacchetti De Gregoris et al., 2011). Reaction mixtures contained 10 µL of SYBR Green PCR Realtime PCR Master mix (Toyobo, Japan), 100 nmol of each primer, 1 µL of DNA template (The total DNA), and distilled water to a final volume of 20 µL. The amplification was carried out using a protocol by Bacchetti De Gregoris et al. (2011) with the modification of annealing temperature as follows: initial denaturation at 50°C for 2 min; 95°C for 10 min; followed by 40 cycles at 95°C for 15 s, 65°C for 30 s, and 72°C for 15 s, and a final extension at 40°C for 420 s. The plasmid DNA from the positive clone, obtained from the 16S rRNA library, was used as the standard for qPCR assay. Standard curves were set up using 10-fold serial dilutions of the plasmid containing a partial sequence of the Actinobacteria 16S rRNA gene, obtained from the 16S rRNA library using Mini Plasmid Kit (Tiangen, Beijing, China). The copy number of the target gene was calculated based on the concentration of plasmid DNA and its molecular weight. Plasmid DNA concentration was determined on a spectrophotometer (Nanodrop Technologies, Wilmington, USA).
qPCR run was completed with a melting analysis (60 – 95°C, ramp 0.5°C min−1) to check for product specificity and primer dimmers formation. All samples and standards were run in triplicates. Amplification efficiencies of the PCR reactions were calculated using data from the standard curves with the formula Eq. 4 (Cikos and Koppel 2009). The quality of the amplification was evaluated by the generation of melting curves of the PCR products.
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Libraries construction and amplified rRNA gene restriction analysis In this study, to reveal the biodiversity of the bacterial communities in the aged and aging pit mud, the total DNA from the aged and aging pit mud samples was used to generate two clone libraries. A total of 172 clones were obtained and studied. The number of positive clones was 87 in the aged pit mud 16S rRNA library and was 85 in aging pit mud 16S rRNA library, respectively (Table 1.).
| Bacterial | ||
|---|---|---|
| Aged pit mud | Aging pit mud | |
| Clones analysed | 85 | 87 |
| OTUs determined | 20 | 9 |
| Affiliated taxa | 10 | 7 |
| Coverage | 96.6% | 96.5% |
| Diversity index | 2.48 | 1.85 |
| Evenness | 0.83 | 0.80 |
To avoid sequencing clones with identical 16S rRNA genes, the insert-containing plasmids were digested with the restriction enzyme Hha I to generate amplified rRNA gene restriction analysis patterns. OTU was defined as a unique amplified rRNA gene restriction analysis pattern. A total of 23 different patterns were generated and sequenced. Table 1 showed the number of clones analyzed for each library, the OTUs and number of ascertained taxa. The Shannon diversity index of each library ranged from 1.85 to 2.48. It showed that the bacterial diversity of the aged pit mud was higher than that of the aging pit mud.
The rarefaction curves from the bacterial libraries were respectively obtained by plotting the number of OTUs observed against the number of clones (Fig. 1.). The decrease in the rate of OTUs detection shown on the curve indicated that the major part of the diversity in pit mud 16S rRNA libraries had been detected. This conclusion was further supported by calculating the coverage of two libraries. The coverage ranged from 96.6% to 96.5%, which suggested that the number of analyzed OTUs should be satisfactory and that most bacteria present in the samples detected. Fig. 2. showed the difference of OTUs between the aged and aging pit mud and the different bacterial structure between the aged and aging pit mud. It was obvious that the Actinobacteria community was only detected in the aged pit mud.
Rarefaction analysis of pit mud bacterial 16S rRNA libraries, displaying the number of clones studied versus the number of OTUs detected. Aged pit mud and aging pit mud respectively represented 16S rRNA libraries of aged and aging pit mud.
Distribution of the bacterial clones in the aged pit mud and aging pit mud libraries.
16S rRNA phylogenetic analysis A phylogenetic analysis of the bacteria 16S rRNA sequences from the clone libraries revealed that the bacterial difference between the aged and aging pit mud was obvious. The bacterial abundance of the aged pit mud was higher than that of the aging pit mud. The Firmicutes and Bacteroidetes phyla were identified both in the aged and aging pit mud clone library, whereas Chloroflexi and Actinobacteria were only detected in the aged pit mud.
Firmicutes is well-known to be syntrophic bacteria that can degrade volatile fatty acids, such as butyrate and its analogs (Garcia-Peña et al., 2011). Firmicutes is represented by the Clostridia and Bicilli classes. The OTUs affiliated with the Clostridia were represented in both of the samples and accounted for 51.7% of the aged pit mud clones and 67.1% of the aging pit mud (Fig. 3.). It has been reported that Clostridiales family can use ethanol and acetic acid to produce acetoacetate (Kencaly et al., 1995). Some Clostridium species (in the class Clostridia), such as C. sticklandii and C. aminobutyricum, were reported as microorganisms capable of utilizing amino acids and producing acetate, ammonia, and butyrate (Shin et al., 2010). Therefore, high levels of Clostridiales in pit mud might lead to the reduction of acetoacetate whose synthesis requires ethanol and acetic acid. The only OTU affiliated with Firmicutes, Thermoanaerobacteraceae genera was detected in the aged pit mud. Kaksonen et al. (2006) reported that Thermoanaerobacteraceae was able to reduce SO42− to S2− and Fe3+ to Fe2+. Thus, insoluble ferrous sulfides were produced, which reduced the production of ferrous lactate so that the aging process of pit mud might be prevented.
Phylogenetic tree of the detected OTUs from the phylum bacteriophyta of the domain bacteria in the aged and aging pit mud based on neighbor-joining analysis of partial 16S rRNA nucleotide sequences. The genetic distances were computed using the Jukes-Cantor method and are represented by the units of the number of base substitutions per site. The numbers in brackets behind the cloning landing represents the number of clones of the aged and aging pit mud clone library, respectively.
Species from the Bacteroidetes phylum are acidogenic bacteria that produce propionate, acetate and succinate (Cardinali-Rezende et al., 2012). The Bacteroidetes phylum accounted for 2.3% of the sequences in the aged pit mud and 31.8% in the aging pit mud, respectively. Therefore, the excessive presence of Bacteroidetes might produce salt compounds that made pit mud harden. That was why Bacteroidetes in the aging pit mud was more abundant than in the aged pit mud.
Chloroflexi species could take up butyrate and pyruvate (Kragelund et al., 2007). In the aged pit mud, the only OTU affiliated with Anaerolineaceae in Chloroflexi phylum was detected. Kragelund et al. (2007) found that the family Anaerolineaceae predominated in the deep layers which were in agreement with the result in this paper. What is more, Anaerolineaceae strains could produce H2 and had syntrophy with methanogens (Yamada et al., 2009).
Actionbacteria has positive effect on the pit mud (Zhang et al., 2006, Jiang et al., 2008) and at present, the role of Choroflexi in pit mud is unclear. Naturally, the study of Choroflexi is in progress conducted by our group. Therefore, this study originally researched the number of Actionbacteria in the aged and aging pit mud. Actinobacteria was able to utilize sulfides which resulted in the aging of pit mud and could ferment with caproic acid bacteria so as to improve the yield of caproic acid contributing to the aroma of wine (Chao et al., 2011). Meanwhile, Zhang et al. (2006) found that the number of Actinobacteria increased in the aged pit mud which was in accordance with the result in this study.
Quantitative analysis of Actinobacteria using qPCR To analyze the bacterial diversity of the aged and aging pit mud more exactly, the Actinobacteria genus of the aged and aging pit mud was monitored using qPCR. The amplification curves and melting curves of the Actinobacteria in pit mud were shown in Fig. 4. It showed that the melting curves were smooth and each melting curve had only a peak. It indicated that the specificity of primers which were used to quantify Actinobacteria were good. The formula of Actinobacteria using qPCR was as Eq. 5.
Amplification curves (a) and melting curves (b) of Actinobacteria in pit mud. A and B represent the amplification curves of the aged pit mud and the aging pit mud, respectively.
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The slope of the qPCR assays was −3.45. The amplification efficiency was 95% with R2 > 0.99. The results indicated that qPCR had good amplification efficiency. It was shown for Actinobacteria to exist at a (1.68 ± 0.04) × 1010 copies per g of pit mud in the aged pit mud and at a (0.58 ± 0.01) × 109 copies per g of pit mud in the aging pit mud. It suggested that the quantity of Actinobacteria genus in the aged pit mud should be 29 times as much as in the aging pit mud (P < 0.05).
Meanwhile, Zhang et al. (2006) found that the number of Actinobacteria in good quality pit mud (2.6 × 106 cells per g of pit mud) was more than that in bad quality pit mud (7.4 × 104 cells per g of pit mud) using culture-dependent methods, which was in accordance with the result of this study. However, qPCR is more convenient and effective than culture-dependent methods. Therefore, the number of Actinobacteria might be the sign of the aged pit mud. qPCR has gained wide acceptance due to its improved rapidity, sensitivity, reproducibility and reduced risk of carry-over contamination (Mackay et al., 2002). Moreover, the qPCR assay for detecting microorganisms in pit mud has not been reported. In this paper, qPCR analyses effectively distinguished the aged pit mud from the aging pit mud.
The result of qPCR was confirmed that the number of Actinobacteria in aged pit mud was more than that in aging pit mud from other Luzhou-flavor liquor distillery using qPCR analysis. For the 16S rRNA gene clone libraries analysis, further researches are in progress conducted by our laboratory group.
In the present study of pit mud, there are many reports of pit mud in Luzhou-flavor liquor distillery. For example, Jiang et al. (2008) and Zhang et al. (2006) compared the differences of physical and chemical properties between the normal pit mud and the aging pit mud based on culture-dependent methods, which is difficult to reveal the inner pattern comprehensively and objectively. Deng et al. (2012) also analyzed the differences in pit mud of cellars used for different periods of time while there was no information about the bacteria gene sequence. Moreover, Shi et al. (2012) reported the bacterial diversity of pit mud from different ages. However, there was litter report about the biodiversity of the bacterial communities in aged pit mud and aging pit mud. This study found that Firmicutes and Bacteroidetes phyla were identified both in the aged and aging pit mud and Chloroflexi and Actinobacteria were more abundant in the aged pit mud using 16S rRNA gene clone libraries analysis and qPCR analysis which is rapid and accurate.
This study has revealed the rich diversity of bacterial community in the aged and aging pit mud for the first time. The 16S rRNA gene clone libraries analysis could obtain general information of microorganisms in pit mud and the qPCR analysis is rapid, sensitive and accurate. Consequently the combination of the 16S rRNA gene clone libraries and qPCR analysis could be better to reveal the microbial information in the environment. The information obtained may be useful to improve the understanding of the composition of the microbes of Luzhou-flavor liquor, to reveal the mechanism from aged pit mud to aging pit mud and to carry out the effective management for improving the quality and yield of Luzhou-flavor liquor.
Acknowledgements This project was financially supported by the National Natural Science Foundation of China (No. 31071585).
