Landoltia punctata, a widely distributed duckweed strain with the ability to accumulate starch, was used as a novel feedstock for bioethanol production by Saccharomyces cerevisiae. To improve ethanol production, pectinase pretreatment was used to release much more glucose from L. punctata mash and the pretreatment conditions (enzyme loading, temperature and pretreatment time) for the duckweed were optimized by using a surface response design. The results showed that maximum glucose yield was 218.64 ± 3.10 mg/g dry matter, which is a 142% increase compared to the untreated mash, with a pectinase dose of 26.54 pectin transeliminase unit/g mash at 45 °C for 300 min. Pectinase pretreatment apparently changed the ultrastructure of L. punctata, as evidenced by scanning electron microscopy analysis. Further fermentation experiments were performed and 30.8 ± 0.8 g/L of ethanol concentration, 90.04% of fermentation efficiency and 2.20 g/L^/h of productivity rate were achieved. This is the highest ethanol concentration reported to date using duckweed as the feedstock.

Background: Duckweed 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. Results: This 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. Conclusion: Our 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.

Landoltia punctata is a widely distributed duckweed species with great potential to accumulate enormous amounts of starch for bioethanol production. We found that L. punctata can accumulate starch rapidly accompanied by alterations in endogenous hormone levels after uniconazole application, but the relationship between endogenous hormones and starch accumulation is still unclear. After spraying fronds with 800 mg/L uniconazole, L. punctata can accumulate starch quickly, with a dry weight starch content of up to 48% after 240 h of growth compared to 15.7% in the control group. Electron microscopy showed that the starch granule content was elevated after uniconazole application. The activities of key enzymes involved in starch synthesis were also significantly increased. Moreover, the expression of regulatory elements of the cytokinin (CK), abscisic acid (ABA) and gibberellin (GA) signaling pathways that are involved in chlorophyll and starch metabolism also changed correspondingly. Importantly, the expression levels of key enzymes involved in starch biosynthesis were up-regulated, while transcript-encoding enzymes involved in starch degradation and other carbohydrate metabolic branches were down-regulated. The increase of endogenous ABA and CK levels positively promoted the activity of ADP-glucose pyrophosphorylase (AGPase) and chlorophyll content, while the decrease in endogenous GA levels inactivated α-amylase. Thus, the alterations of endogenous hormone levels resulted in starch accumulation due to regulation of the expression of genes involved in the starch metabolism pathway.