Kaize He

Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation

BackgroundDuckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed.ResultsThis study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples.ConclusionOur study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata.

Microbial community and removal of nitrogen via the addition of a carrier in a pilot-scale duckweed-based wastewater treatment system

Carriers were added to a pilot-scale duckweed-based (Lemna japonica 0223) wastewater treatment system to immobilize and enhance microorganisms. This system and another parallel duckweed system without carriers were operated for 1.5 years. The results indicated the addition of the carrier did not significantly affect the growth and composition of duckweed, the recovery of total nitrogen (TN), total phosphorus (TP) and CO2 or the removal of TP. However, it significantly improved the removal efficiency of TN and NH4(+)-N (by 19.97% and 15.02%, respectively). The use of 454 pyrosequencing revealed large differences of the microbial communities between the different components within a system and similarities within the same components between the two systems. The carrier biofilm had the highest bacterial diversity and relative abundance of nitrifying bacteria (3%) and denitrifying bacteria (24% of Rhodocyclaceae), which improved nitrogen removal of the system. An efficient N-removal duckweed system with enhanced microorganisms was established.

Using proteomic analysis to investigate uniconazole-induced phytohormone variation and starch accumulation in duckweed (Landoltia punctata).

BackgroundDuckweed (Landoltia punctata) has the potential to remediate wastewater and accumulate enormous amounts of starch for bioethanol production. Using systematical screening, we determined that the highest biomass and starch percentage of duckweed was obtained after uniconazole application. Uniconazole contributes to starch accumulation of duckweed, but the molecular mechanism is still unclear.ResultsTo elucidate the mechanisms of high starch accumulation, in the study, the responses of L. punctata to uniconazole were investigated using a quantitative proteomic approach combined with physiological and biochemical analysis. A total of 3327 proteins were identified. Among these identified proteins, a large number of enzymes involved in endogenous hormone synthetic and starch metabolic pathways were affected. Notably, most of the enzymes involved in abscisic acid (ABA) biosynthesis showed up-regulated expression, which was consistent with the content variation. The increased endogenous ABA may up-regulate expression of ADP-glucose pyrophosphorylase to promote starch biosynthesis. Importantly, the expression levels of several key enzymes in the starch biosynthetic pathway were up-regulated, which supported the enzymatic assay results and may explain why there is increased starch accumulation.ConclusionsThese generated data linked uniconazole with changes in expression of enzymes involved in hormone biosynthesis and starch metabolic pathways and elucidated the effect of hormones on starch accumulation. Thus, this study not only provided insights into the molecular mechanisms of uniconazole-induced hormone variation and starch accumulation but also highlighted the potential for duckweed to be feedstock for biofuel as well as for sewage treatment.

New microbial resource: microbial diversity, function and dynamics in Chinese liquor starter

Traditional Chinese liquor (Baijiu) solid state fermentation technology has lasted for several thousand years. The microbial communities that enrich in liquor starter are important for fermentation. However, the microbial communities are still under-characterized. In this study, 454 pyrosequencing technology was applied to comprehensively analyze the microbial diversity, function and dynamics of two most-consumed liquor starters (Jiang- and Nong-flavor) during production. In total, 315 and 83 bacterial genera and 72 and 47 fungal genera were identified in Jiang- and Nong-flavor liquor starter, respectively. The relatively high diversity was observed when the temperature increased to 70 and 62 °C for Jiang- and Nong-flavor liquor starter, respectively. Some thermophilic fungi have already been isolated. Microbial communities that might contribute to ethanol fermentation, saccharification and flavor development were identified and shown to be core communities in correlation-based network analysis. The predictively functional profile of bacterial communities showed significant difference in energy, carbohydrate and amino acid metabolism and the degradation of aromatic compounds between the two kinds of liquor starters. Here we report these liquor starters as a new functionally microbial resource, which can be used for discovering thermophilic and aerobic enzymes and for food and feed preservation.

Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter

Chinese liquor is one of the worlds best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.

Responses of Landoltia punctata to cobalt and nickel: Removal, growth, photosynthesis, antioxidant system and starch metabolism

Landoltia punctata has been considered as a potential bioenergy crop due to its high biomass and starch yields in different cultivations. Cobalt and nickel are known to induce starch accumulation in duckweed. We monitored the growth rate, net photosynthesis rate, total chlorophyll content, Rubisco activity, Co2+ and Ni2+ contents, activity of antioxidant enzymes, starch content and activity of related enzymes under various concentrations of cobalt and nickel. The results indicate that Co2+ and Ni2+ (≤0.5mgL-1) can facilitate growth in the beginning. Although the growth rate, net photosynthesis rate, chlorophyll content and Rubisco activity were significantly inhibited at higher concentrations (5mgL-1), the starch content increased sharply up to 53.3% dry weight (DW) in L. punctata. These results were attributed to the increase in adenosine diphosphate-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSS) activities and the decrease in α-amylase activity upon exposure to excess Co2+ and Ni2+. In addition, a substantial increase in the antioxidant enzyme activities and high flavonoid contents in L. punctata may have largely resulted in the metal tolerance. Furthermore, the high Co2+ and Ni2+ contents (2012.9±18.8 and 1997.7±29.2mgkg-1 DW) in the tissue indicate that L. punctata is a hyperaccumulator. Thus, L. punctata can be considered as a potential candidate for the simultaneous bioremediation of Co2+- and Ni2+-polluted water and high-quality biomass production.

Combination of RNA sequencing and metabolite data to elucidate improved toxic compound tolerance and butanol fermentation of Clostridium acetobutylicum from wheat straw hydrolysate by supplying sodium sulfide

Sodium sulfide (SS) was added to the non-detoxified wheat straw hydrolysate for ABE fermentation by Clostridium acetobutylicum CICC8012. Biochemical measurements demonstrated that supplementation with SS promoted earlier and enhanced conversion of acid to ABE and led to a 27.48% improvement in sugar consumption, a 20.48% improvement in the sugar-based ABE yield, a 47.63% improvement in the butanol titer, and a 53.50% improvement in the ABE concentration. The response of C. acetobutylicum CICC8012 at the mRNA level was examined by a transcriptional analysis performed with RNA sequencing. The expression of genes involved in the membrane transport of carbohydrates, glycolysis, and ABE formation increased following SS-supplemented fermentation, whereas the expression of genes encoding enzymes involved in acid formation decreased, which indicates that supplemental SS affected the central fermentative pathway, down-regulated the metabolic flux toward the acid formation branches, and up-regulated the metabolic flux toward the ABE formation branches.

Duckweed systems for eutrophic water purification through converting wastewater nutrients to high-starch biomass: comparative evaluation of three different genera ( Spirodela polyrhiza , Lemna minor and Landoltia punctata ) in monoculture or polyculture

The polyculture of different duckweed species is likely to integrate their advantages in removing pollutants and starch accumulation. Here, pilot-scale comparisons of three duckweed species (Spirodela polyrhiza K1, Lemna minor K2 and Landoltia punctata K3) in monoculture and polyculture were investigated. Results showed that the TN (total nitrogen) and TP (total phosphorus) in wastewater decreased from 6.0 and 0.56 mg L−1 to below 0.5 and 0.1 mg L−1, respectively. Namely, the water quality improved to Grade II under the Chinese standard. The highest TN and TP removal efficiencies were found to be 99.1% and 90.8% in the polyculture. Besides, the starch content of S. polyrhiza K1, L. minor K2, L. punctata K3 and the polyculture reached 24.8%, 32.3%, 39.3% and 36.3%, respectively. Accordingly, their average starch accumulation rates were 1.65, 2.15, 3.11 and 2.72 g m−2 d−1, respectively. Our results suggested that L. punctata K3 was a promising energy feedstock due to it having the highest starch production. The advantages of different duckweed species were investigated. In the polyculture, the pollutants were efficiently removed from wastewater, with a high starch accumulation. This study supplies a new insight into the application of duckweed in eutrophic water advanced treatment coupled with starch production.

Transcriptome analysis of Saccharomyces cerevisiae at the late stage of very high gravity (VHG) fermentation

2 (T1), algae plus commercial feed at the stocking rate of 10 fishes/m 2 (T2), and commercial feed only at the stocking rate of 20 fishes/m 2 (T3). The tanks were stocked with O. niloticus fingerlings with mean weight of 1.160, 1.030 and 0.255 g for T1, T2 and T3, respectively. Each treatment was replicated twice. Data collected were analyzed using analysis of variance and mean comparison was carried out using Duncan Multiple Range Test (DMRT). All tests were carried out at 5% probability level. The result on growth performance show that the intensive system (T1) was significantly different in some growth parameters (P < 0.05) such as yield (0.115, 0.830 and 1.260 kg/m 3 for T1, T2 and T3, respectively), specific growth (1.975, 2.750 and 3.425% for T1, T2 and T3, respectively), survival rate (90, 80 and 91% for T1, T2 and T3, respectively), and feed conversion ratio (1.150 and1.030 for T2 and T3, respectively). Economically, the intensive system was more profitable with a gross margin of N 122, which was 40% more profitable than the extensive system (T1 with Gross margin of N 80), and 269% than commercial feed plus algae (T2 with Gross margin of - N 194). The cost of production (incidence of cost) showed that it cost more (N 447) to produce O. niloticus using commercial feed plus algae (T2 ) in concrete tank than the use of algae only (T1) (N 268) and the use of commercial feed only (T3) (N380).

Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-β-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-β-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60 °C and highly stable (>75% residual activity) at pH 3-8 and temperature 30-90 °C. The activity of NFEg16A was strongly inhibited by 10 mM Fe3+ and Hg2+. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70 °C and had higher half-lives of 3.4 h and 1.4 h at 80 °C and 90 °C, respectively. Its specific activity was 85.3 U/mg on barley β-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.