Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, β-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis.

Bacterial communities in high-temperature Daqu and fermented grains are important for brewing Jiang-flavor Baijiu such as Danquan Baijiu. Daqu is a saccharifying and fermenting agent, which has a significant impact on the flavor of Baijiu. However, bacterial communities in three different types of samples from the Danquan distillery (dqjq_ck, dqjqcp, and dqjp3) were still unclear, which limited further development of Danquan Baijiu. “dqjq_ck” and “dqjqcp” indicate high-temperature Daqu at days 45 and 135, respectively. “dqjp3” indicates fermented grains. In this study, the bacterial communities of three samples were analyzed by Illumina Miseq high-throughput sequencing. The bacterial communities of three samples primarily composed of thermophilic bacteria and bacteria with stress resistance. The most abundant species in dqjq_ck, dqjqcp, and dqjp3 were Comamonas, Bacillus, and unclassified Lactobacillales, respectively. The main bacteria included Bacillus, Comamonas, Myroides, Paenibacillus, Acetobacter, Kroppenstedtia, Staphylococcus, Saccharopolyspora, Planifilum, Lactobacillus, Acinetobacter, Oceanobacillus, Enterococcus, Thermoactinomyces, Lactococcus, Streptomyces, Saccharomonospora, Tepidimicrobium, Anaerosalibacter, unclassified_Lactobacillales, unclassified_Thermoactinomycetaceae_1, unclassified_Bacillaceae_2, unclassified_Bacillales, unclassified_Microbacteriaceae, unclassified_Rhodobacteraceae, unclassified_Actinopolysporineae, and unclassified_Flavobacteriaceae in three samples (percentage was more than 1% in one of three samples). In our study, the succession of microbiota in three samples representing three important stages of Danquan Baijiu brewing was revealed. This article lays a good foundation for understanding the fermentation mechanism and screening some excellent indigenous bacteria to improve the quality of Danquan Baijiu in future.