Jiang-flavor (JF) daqu is a liquor starter used for production of JF baijiu, a well-known distilled liquor in China. Although a high temperature stage (70°C) is necessary for qualifying JF daqu, little is known regarding its active microbial community and functional enzymes, along with its role in generating flavor precursors for JF baijiu aroma. In this investigation, based on metatranscriptomics, fungi, such as Aspergillus and Penicillium, were identified as the most active microbial members and 230 carbohydrate-active enzymes were identified as potential saccharifying enzymes at 70°C of JF daqu. Notably, most of enzymes in identified carbohydrate and energy pathways showed lower expression levels at 70°C of JF daqu than those at the high temperature stage (62°C) of Nong-flavor (NF) daqu, indicating lowering capacities of saccharification and fermentation by high temperature stage. Moreover, many enzymes, especially those related to the degradation of aromatic compounds, were only detected with low expression levels at 70°C of JF daqu albeit not at 62°C of NF daqu, indicating enhancing capacities of generating special trace aroma compounds in JF daqu by high temperature stage. Additionally, most of enzymes related to those capacities were highly expressed at 70°C by fungal genus of Aspergillus, Coccidioides, Paracoccidioides, Penicillium, and Rasamsonia. Therefore, this study not only sheds light on the crucial functions of high temperature stage but also paves the way to improve the quality of JF baijiu and provide active community and functional enzymes for other fermentation industries.

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Background Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. Results In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. Conclusions This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

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A B S T R A C TThe duckweed Lemna aequinoctialis was used as a biosorbent material for Cd 2+ adsorption in this study. Influencing factors of Cd 2+ adsorption by L. aequinoctialis in aqueous solution were investigated and the process of the Cd 2+ biosorption was optimized. The results of single-factor experiments suggested that all the factors studied except temperature had significant effects on the removal efficiency of Cd 2+ by L. aequinoctialis. Based on the results of single-factor experiments, optimization of the Cd 2+ biosorption was performed by varying four independent parameters using the central composite design under response surface methodology. The optimal conditions for the maximum removal of Cd 2+ were as follows: grain size of 150-200 mesh, stirring speed of 75 rpm, Cd 2+ initial concentration of 40 mg/L, and sorbent concentration of 8 g dry matter/L. The maximum removal efficiency of 83.5% was obtained, which was in consistence with the predicted value of 83.6%. This process followed pseudo-second-order kinetics and the experimental data fitted well to Langmuir and Freundlich isotherm models. The maximum capacity of duckweed to adsorb Cd 2+ was 33.0 mg/g, demonstrating that untreated dry powder of L. aequinoctialis represents a promising biosorbent for Cd 2+ removal. Fourier transform infrared spectroscopy analysis indicated that the -OH groups of carbohydrate compounds and the -NH 2 groups of amide compounds may be the main groups involved in the adsorption of Cd 2+ by L. aequinoctialis.

Terrestialization is supposedly an important evolutionary process plant experience. However, directions of land back to water acquired little attention. Here we integrate multiproxy evidence to elucidate the evolution of duckweed. Three genera of duckweed show chronologically gradient degeneration in roots structure and stomatal function and decrease in lignocellulose content, accompanied by gradual contraction in relevant gene numbers and/or decline in transcription. The gene numbers in the main phytohormonal pathway are also gradually decreased. The co-action of auxin and rhizoid development gene causes a gradual decrease in adventitious roots. The significant expansion of the flavonoid pathway is also related to the adaptation of duckweed to floating growth. This study reconstructs the evolution history of duckweeds from land back to water, reverse to that of early land plants.

In order to develop efficient biosorbent material for Pb 2+ removal from wastewater, this study explored the biosorption of duckweed (Lemna aequinoctialis) powder. Single-factor experiments were designed to investigate the dominant effect on Pb 2+ removal. The experimental data were fitted with adsorption kinetics and isotherms to investigate the adsorption properties. The infrared spectroscopy was used to probe the surface characteristics of the sorbent. The results of single-factor experiments suggested that initial pH, grain size of sorbent and initial concentration of Pb 2+ had significant effects on the removal efficiency of Pb 2+ . The infrared spectroscopy analysis indicated that the -OH groups of carbohydrate compounds and the -NH 2 groups of amide compounds may be the main groups involved in the adsorption of Pb 2+ by the powder of L. aequinoctialis. This process followed the pseudo second-order kinetic with a correlation coefficient (R 2 ) of 0.999 95 and the k 2 of 3.65 g min -1 mg -1 . The experimental data were well fitted with Freundlich isotherm model and the maximum Pb 2+ adsorption capacity was higher than 56.79 mg/g. This study suggested that the powder of L. aequinoctialis, as a new biosorbent material, has high Pb 2+ adsorption rate and capacity under various conditions. Fig 5, Tab 2, Ref 31

The fungal community in Qu plays a key role in the formation of turbid rice wine (TRW) style. The Sichuan Basin and its surrounding areas have become one of the main TRW production regions in China; however, the fungal community in Qu and how they affect the characteristics of TRW remain unknown. Therefore, this study provided insight into the fungal biomarkers in Qu from Guang’an (GQ), Dazhou (DQ), Aba (AQ), and Liangshan (LQ), as well as their relationships with compounds in TRW. The main biomarkers in GQ were Rhizopus arrhizus, Candida glabrata, Rhizomucor pusillus, Thermomyces lanuginosus and Wallemia sebi. However, they changed to Saccharomycopsis fibuligera and Mucor indicus in DQ, Lichtheimia ramose in AQ, and Rhizopus microsporus and Saccharomyces cerevisiae in LQ. As a response to fungal biomarkers, the reducing sugar, ethanol, organic acids, and volatile compounds were also changed markedly in TRWs. Among important volatile compounds (VIP > 1.00), phenethyl alcohol (14.1–29.4%) was dominant in TRWs. Meanwhile, 3-methyl-1-butanol (20.6–56.5%) was dominant in all TRWs except that fermented by GQ (GW). Acetic acid (29.4%) and ethyl palmitate (10.1%) were dominant in GW and LW, respectively. Moreover, GQ biomarkers were positively correlated with acetic acid and all unique important volatile compounds in GW. DQ biomarkers had positive correlations with unique compounds of acetoin and ethyl 5-chloro-1,3,4-thiadiazole-2-carboxylate in DW. Meanwhile, the AQ biomarkers were positively correlated with all AW unique, important, and volatile compounds. Although there were not any unique volatile compounds in LW, 16 important volatile compounds in LW were positively related to LQ biomarkers. Obviously, biomarkers in different geographic Qu played vital roles in the formation of important volatile compounds, which could contribute specific flavor to TRWs. This study provided a scientific understanding for future efforts to promote the excellent characteristics of TRW by regulating beneficial fungal communities.