Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people’s health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function. IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

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Two bacterial strains, paraffin removal strain and biosurfactant-producing strain, named BHJ-1 and QFL-1, were isolated from oil production wells in Daqing oilfield of China. They were subsequently identified as Bacillus cereus QAU68 and Bacillus subtilis XCCX, respectively. As an indicator of the degradation paraffin, the inoculum concentration of BHJ-1 and QFL-1 were added in different proportions, the optimum proportion was 5:2. In this proportion the degradation rate of paraffin could reach 64 %, the prevention rate of paraffin could reach 55 %.Keywords Paraffin Á Bacillus cereus Á Bacillus subtilis Á Degradation Microbial treatment method of paraffin deposition has been used as a sustainable replacement to conventional treatment methods (chemical, mechanical and thermal methods) [1,2]. Industrial scale tests were accomplished in Jidong, Daqing, Liaohe and Zhongyuan oilfields in China [3], but there is no large-scale application by microbial treatment on the whole, the reasons for restricting the large-scale application are short of validity period and less efficiency. Adding special biosurfactant can lower the interfacial tension as their primary activity [4,5], increase the hydrocarbon concentration in the water [6,7], therefore adding the special biosurfactant may be propitious for combination of crude oil and bacterial liquid, prolong the validity period, increase the efficiency of microbial treatment and paraffin deposition.During this study two isolates, paraffin removal strain and biosurfactant-producing strain, named BHJ-1 and QFL-1, were isolated from oil production wells in Daqing Oilfield of China. BHJ-1 was isolated from enrichment salts medium (ESM) containing 0.5 % (w/v) of crude oil. The system was operated at the following parameters: temperature 37°C, pH 7.2, incubation period 7 days. In order to isolate oil-utilizing microorganisms, 0.1 ml of culture supernatant was transferred every 24 h onto nutrient agar plates [8]. QFL-1 was isolated from ESM and it was cultivated at orbital shaking (180 rpm) at 37°C for 72 h. The culture fluid was spread on the cetyl tri ammonium bromide-methylene blue agar medium and cultivated at 37°C for 9 days. Then, the colonies of the halo were extracted, streaked on crude oil agar medium and cultivated at 37°C for 9 days. After five times plate streak, the pure strains could be purified [9]. The isolates were stored in isosensitest broth at 4°C and were plated in Isosensitest agar for further investigation [10]. The photo of scanning electron microscope is shown in Fig. 1. Phylogenetic tree was constructed and analysis was done by neighbour joining method through MEGA 5 software (Fig. 2). Partial sequencing with both primers revealed that the closest matches, determined by a BLAST search, corresponded to Bacillus cereus QAU68 (99 % similarity, BHJ-1) and Bacillus subtilis XCCX (99 % similarity, QFL-1), respectively [11,12].As an indicator of the degradation of paraffin, BHJ-1 and QFL-1 were added in different proportions. The

Tyrosine crystals occasionally appeared on the broad bean paste surface, which will cause economic losses. This study aimed to eliminate the tyrosine crystals in broad bean paste through decreasing the activities of proteolytic enzymes produced by Aspergillus oryzae and process optimization. Broad bean pastes containing no more than 6.16 mg/g dry material tyrosine showed low possibility to form tyrosine crystals. Using tyrosine as substrate, the A. oryzae 3.042 was adaptively evolved and the tyrosine content in the broad bean paste fermented by the evolved A. oryzae was reduced from 6.49 mg/g dry material to 6.14 mg/g dry material (p < 0.05). When the production process was optimized, the tyrosine content in broad bean paste was further reduced to 5.67 mg/g dry material (p < 0.05). In this condition, no tyrosine crystals were formed in broad bean paste after the 12‐month storage while the product quality was not influenced. Practical applications Tyrosine crystals were one of the most important factors which negatively influence the quality of traditionally fermented food, including broad bean paste, soybean paste, and sausages. The appearance of tyrosine crystals in these foods will cause economic losses to manufacturers. This study tried to eliminate the appearance of tyrosine crystals through decreasing the activities of proteolytic enzymes produced by Aspergillus oryzae 3.042 and fermentation process optimization. The adoption of modified A. oryzae and optimized fermentation process successfully guaranteed the elimination of tyrosine crystals formation in the production and storage period of broad bean paste. This will not only benefit the broad bean paste manufacturers but also provide guidance for other fermented food producers to deal with tyrosine crystals problem.

The aim of the study was to increase the production of biosurfactant by using the inexpensive medium materials and the optimized fermentation conditions. Five biosurfactant producers were isolated from oilfield wastewater. The most efficient strain S2 was identified through 16S rDNA sequence analysis, which exhibitted the highest similarities to Bacillus subtilis strains CCTCC M201162. The optimal medium composition of this strain were beer wastewater 40 g/L, diammonium phosphate (NH 4 H 2 PO 4 ) 4 g/L, MgSO 4 0.02 g/L, NaCl 5 g/L. The emulsification index (E 24 ) was optimized using a box-behnken experimental design and response surface methodology(RSM). The RSM revealed that when pH was 7.2, temperature was 42.1 C, inoculum concentration was 5.2% (v/v) and rotate speed was 163 r/min, the optimal condition was obtained. Under this condition, the optimal E 24 of the biosurfactant produced by Bacillus subtilis strains CCTCC M201162 was 81.20%, the production of crude biosurfactant increased from 0.72 g/L to 1.26 g/L and the surface tension of fermentation broth reduced from 72.0 mN/m to 22.8 mN/m. A quadratic response model was constructed through RSM designs, leading to a 75.0% increase of biosurfactant production by Bacillus subtilis strains CCTCC

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Microorganisms were screened from oil wastewater in Daqing Oilfield, by acclimatization, isolation and purification of microorganisms, bacterium as the only nitrogen source was obtained, which was named for strain Y-1. Experiment of identification showed that Y-1 was identified to Bacillus Cereus. Strain Y-1 was used for paraffin removal on three wells: Well Nos. 12-36, 13-39 and 14-43 in Yushulin Zone Daqing Oilfield. The results indicated that the tested wells obtained good effects after microbial treatment: the reduction rate of oil viscosity was 23.6%, the freezing point of crude oil reduced by 2.1°C, the reduction rate of oil surface tension was 38.0%, pH value was reduced from 7.2 to 6.2, daily oil production increased from 1.7t·d-1 to 2.4t·d-1, the rate of growth was 41.2%, the period of hot washings prolonged from 40d to 149d and the number of hot washings reduced by 4 times in Well 12-36; the reduction rate of oil viscosity was 24.8%, the freezing point of crude oil reduced by 2.7°C, the reduction rate of oil surface tension was 42.5%, pH value was reduced from 7.2 to 6.8, daily oil production increased from 1.2t·d-1 to 1.6t·d-1, the rate of growth was 33.3%, the period of hot washings prolonged from 45d to 158d and the number of hot washings reduced by 5 times in Well 13-39; the reduction rate of oil viscosity was 22.5%, the freezing point of crude oil reduced by 3.2°C, the reduction rate of oil surface tension was 42.5%, pH value was reduced from 7.2 to 6.4, daily oil production increased from 0.8t·d-1 to 1.1t·d-1, the rate of growth was 37.5%, the period of hot washings prolonged from 30d to 122d and the number of hot washings reduced by 5 times in Well 14-43. The direct economic income exceeded 210,000 Yuan.

By separation and purification experiment, microorganisms were obtained. The paraffin removal strain was named for SW-1. Microscopic and morphological examinations showed strain SW-1 was to be Bacillus Cereus. After strain SW-1 treatment, the number of strain SW-1 increased from 2.7×106/mL-1to 3.5×108/mL-1, which increased by two orders of magnitude; the degradation rate of paraffin could reach to 64% within one month; the prevention rate of paraffin could reach to 43%; the viscosity of crude oil reduced from 35.9mPa•s to 26.8mPa•s, the reduction rate of viscosity was 25.3%, and the freezing point of crude oil reduced by 4.3°C; surface tension reduced from 44.5mN•m-1to 29.1mN•m-1, the reduction rate of surface tension was 34.6%, and pH value was reduced to 6.3. These phenomenon indicated that strain SW-1 has better removal paraffins effect.