For enrichment and isolation of cultures the following media was used (per litre): glycerol, 20.0 g; (NH₄)₂SO₄, 2.0 g; K₂HPO₄, 3.4 g; KH₂PO₄, 1.3 g; MgSO₄, 0.2 g; CaCl₂, 2 × 10⁻³ g; FeSO₄, 2 × 10⁻³ g; yeast extract powder 1 g; citric acid 0.42 g. Trace element solution 0.2% (v/v) was added to the enrichment media (concentration per liter of deionized water): CoCl₂∙6H₂O 0.2 g; MnCl₂∙4H₂O 0.1 g; ZnCl₂ 0.07 g; H₃BO₃ 0.06 g; Na₂MoO₄・2H₂O 0.035 g; CuCl₂・2H₂O 0.02 g; NiCl₂∙6H₂O 0.025 g. The initial pH at 7.0 regulated by KOH. 18.0 g agar added while the solid medium was needed [15] .

The bottom silt samples were collected from Fenhe River in Shanxi Province of China. Ten grams of silt samples were incubated anaerobically at 37˚C in 300 ml sterile plastic bottles that had been completely filled with enrichment media. After 3 days, Bacteria from each culture were streak-purified on agar plates that were then incubated in (CO₂ Incubator) anaerobic jars with a CO₂ generator (ESCO, America ).

Visual observations of both morphological and microscopic characteristics using the light microscope were conducted. Physiology and biochemistry experiments were determined according to Bergey’s Manual of Determinative Bacteriology [16] . Cell size was observed by scanning electron microscope (SEM) (S570, HITACHI , Japan ).

Used a commercial kit (SK8255, Sangon Biotech), genomic DNA was extracted. A pair of universal primers was prepared according to previously described methods, upstream primer (formerly named 7F ), 5’-CAGAGTTTGATCCTGGCT-3’; downstream primer (named 1540R), 5’-AGGAGGTGATCCAGCCGCA-3’. DNA sequences were determined by Shanghai Sangon Biological Engineering Technology and Service Co. Ltd. ( China ). The closest matches to the partial 16S rDNA sequence were identified based on the results of a BLAST search of the EzBioCloud data-base. The phylogenetic tree was constructed using the neighbor-joining method with the MEGA 5.0 software. Bootstrap analysis for 1000 replicates was performed to estimate the confidence of the tree topologies [17] .

In order to determine the optimum culture environment, the initial concentration of glycerol, the fermentation time and pH value were studied. According to the result of Chao-Ling Wong et al., we set the initial glycerol concentration were 20 g∙L⁻¹, 30 g∙L⁻¹, 40 g∙L⁻¹, 50 g∙L⁻¹, 60 g∙L⁻¹, respectively [5] . Then we take a sample every 6 hours to draw the time curve to determine the optimum fermentation time. Finally, we set the pH gradient was 5 - 10 to determine the optimum fermentation pH.

1,3-PD were analyzed by injecting 0.6 μl of reaction mixture into a gas chromatograph equipped (Aglient GC7820) with a capillary column (ON-Wax, 30 m × 0.32 mm × 0.5 μm). The flow rate of the carrier gas (nitrogen) was 25 ml∙min⁻¹. The column temperature was raised to 180˚C at 15˚C/min and maintained for 10 min, while injector and FID detector temperature were both 250˚C. Standard curve was drawn according to the peak area of 1,3-PD standard substance. The concentration of 1,3-PD standard substance was 2.5 g∙L⁻¹, 5 g∙L⁻¹, 10 g∙L⁻¹, 20 g∙L⁻¹, 40 g∙L⁻¹, respectively. Then using the standard curve (y = 1.5E + 6x + 134372, R² = 0.9993) to calculate the content of 1,3-PD by strain ZH-1.

Residual glycerol was determined by the improved Potassium Permanganate oxidation method according to Wang et al. [18] .

Cell growth was monitored at 650 nm (OD 650) on a spectrophotometer (722S Jinghua , China ).

Of eleven samples collected from the sludge of Fenhe River in China, three strains were able to utilize glycerol growth anaerobically, but only one bacterial strains ZH-1 was able to form higher concentration 1,3-PD. The content of 1,3-PD in the extract can be calculated according to the GC peak area of the 1,3-PD standard substance (Figure 1). Then the Strain ZH-1 was selected for the further studies. By culturing and observed that strain ZH-1 was an aerotolerant anaerobe bacterium, the colonies white mucilaginous, colonies diameter was about 0.6 - 0.8 mm (Figure 2). The SEM studies revealed that strain ZH-1 was Chunky, no flagella bacterium, size 0.5 − 0.6 × 0.9 − 1.4 μm

(Figure 3). The species can be grown on nitrogen-free media, and colonial morphology will vary with different medium components. The physiological and biochemical experiments were shown in Table 1.

We used a 16S rDNA gene sequence-based strategy to identify isolate ZH-1. Full- length (approximately 1.4 kb fragment) 16S rDNA gene was PCR amplified (Figure 4). The complete 16S rDNA of strain ZH-1 (1423 bp) was sequenced and is available at GenBank under accession number KT804692. A BLAST search of EzBioCloud database showed ZH-1 most resembled K. pneumoniae^T strain. The physiological and morphological characteristic shows that the strain ZH-1 was most closely allied to K. pneumoniae. A phylogenetic tree (Figure 5) was constructed based on an alignment of 1423 bp of 16S rDNA sequences.

1,3-PD production depends largely on glycerol―the only carbon and energy source. However, the glycerol tolerance of different strains is inconsistent. Therefore, we first studied the influence of the initial concentration of glycerol (from 20 - 60 g∙L⁻¹) on 1,3-PD production and bacterial biomass at pH 7.0 [5] . As showed in Figure 6, when the glycerol concentration was 20 - 50 g∙L⁻¹, 1,3-PD production and yield increased with the glycerol concentration. However, with the increase of the glycerol concentration from 50 to 60 g∙L⁻¹, the content of 1,3-PD has a slight decline. In addition, Jun et al. [19] reported that 1,3-PD production from Klebsiella pneumoniae DSM 4799 is suppressed

“+” indicates a positive reaction, “−” indicates a negative reaction.

when glycerol concentration was 60 g∙L⁻¹. Wong et al. found that the ability of HE-2 strains in producing 1,3-PD was inhibited when glycerol concentration was 40 - 60 g∙L⁻¹ [5] . In our case, inhibition occurred when the initial glycerol concentration exceeded 50 g∙L⁻¹. When the initial glycerol concentration was 50 g∙L⁻¹, the 1,3-PD yield and biomass reached the maximum, and the molar conversion rate reached 0.4686 mol∙mol⁻¹. Therefore, we determined the optimal initial glycerol concentration was 50 g∙L⁻¹.

Fermentation time plays a critical role in the glycerol transform to 1,3-PD. When the fermentation time is too short, the desired product yield is low that cannot reach the requirements of industrial production. Fermentation time takes too long would make some of the byproducts accumulate and affect the purification of 1,3-PD. To determine optimum fermentation time of K. pneumoniae ZH-1, we carried out batch fermentation research. The initial glycerol concentration was 50 g /L and pH was 7.0, samples were took every 6 hours to detect 1,3-PD concentration, glycerol concentration and biomass, then the fermentation time curve was drawn. As can be seen from the growth curve of bacteria (Figure 7), 0 - 6 h showed a rapid cell growth, 6 - 18 h cells lie in the logarithmic growth phase, after 36 h, cell growth gradually slows down due to the lack of substrate glycerol. In addition, 1,3-PD formation and bacterial growth showed a positive correlation, with the cell reproduction, 1,3-PD have begun to generate, and showed a rapid growth in 0 - 18 h, 1,3-PD production stabilized after 36 h, the amount of residual glycerol was essentially the same after 36 h, so we determine the fermentation time as 36 h.

The changes of pH value have a significant impact on cell growth and product synthesis. Suitable pH value can increase the reaction activity, and accelerate the utilization of

glycerol and increased cell growth and product synthetic rate. In order the maximum production rate of 1,3-PD and the highest bacterial biomass, the pH gradient we selected to study the fermentation optimum pH was 5 to 10. The initial glycerol concentration was 50 g /L and fermentation 36 h, the 1,3-PD production and the bacterial biomass were detected. It was indicated in Figure 8 that the maximum concentration of 1,3-PD was obtained in pH 8.0 media, and the productivity reached 18.53 g∙L⁻¹. At the same time, the molar conversion rate reached 0.497. But when pH was 9.0, the bacterial biomass reached the maximum. We can conclude that it is an alkali-resistant strain. The tolerance to higher pH could probably be related to the genetic characteristics because ZH-1 was isolated from micro-alkaline soil.

After determinations of the glycerol concentration, fermentation time and pH, the glycerol molar conversion rate reached 0.497 mol∙mol⁻¹ from the original 0.34 mol∙mol⁻¹, the molar yield of 1,3-PD to glycerol was consistent with those reported in literatures and those previously reported [7] [20] - [22] . Y. M et al. studied fed-batch of a K. pneumoniae strain on combining biodiesel production by lipase with microbial production of 1,3-PD using a hollow fiber membrane. The molar yield of 1,3-PD to glycerol of 0.47 mol∙mol⁻¹ was obtained [7] . Another study on bioconversion of raw glycerol into 1,3-PD by K. pneumoniae showed that the molar yield of 1,3-PD to glycerol of 0.41 mol∙mol⁻¹ [23] . In this study, the strain ZH-1 had a higher molar conversion rate than some strains of the previous study, whereas compared with the study of Yang et al. (0.62 mol∙mol⁻¹) [24] . The 1,3-PD conversion rate of strain ZH-1 should be further improved, and we will have further research on the strain ZH-1 by adding the metabolites or some other methods.