Spatial Patterns of Planktonic Fungi Indicate Their Potential Contributions to Biological Carbon Pump and Organic Matter Remineralization in the Water Column of South China Sea.

Fungi have long been known to be dynamic in coastal water columns with multiple trophic modes. However, little is known about their interactions with abiotic and biotic components, contribution to the biological carbon pump (BCP), and organic matter remineralization in the oceanic water column. In this study, we investigated how fungi vary spatially and how their variations relate to that of bacteria in the water column of the South China Sea (SCS). Fungi were about three orders less prevalent than bacteria, and the main factors influencing their distribution were depth, temperature, and distance from the sites of riverine inputs. The decline in the abundance of fungi with depth was less steep than that of bacteria. Correlation tests revealed a strong positive association between the abundance of fungi and bacteria, especially in the twilight (r = 0.62) and aphotic (r = 0.70) zones. However, the co-occurrence network revealed mutual exclusion between certain members of fungi and bacteria. The majority of fungi in the water column were saprotrophs, which indicated that they were generally involved in the degradation of organic matter, particularly in twilight and aphotic zones. Similar to bacteria, the involvement of fungi in the metabolism of carbohydrates, proteins, and lipids was predicted, pointing to their participation in the turnover of organic carbon and the biogeochemical cycling of carbon, nitrogen, and sulfur. These findings suggest that fungi play a role in BCP and support their inclusion in marine microbial ecosystem models.

Alpha-Tocopherol Significantly Improved Squalene Production Yield of Aurantiochytrium sp. TWZ-97 through Lowering ROS levels and Up-Regulating Key Genes of Central Carbon Metabolism Pathways.

Media supplementation has proven to be an effective technique for improving byproduct yield during microbial fermentation. This study explored the impact of different concentrations of bioactive compounds, namely alpha-tocopherol, mannitol, melatonin, sesamol, ascorbic acid, and biotin, on the Aurantiochytrium sp. TWZ-97 culture. Our investigation revealed that alpha-tocopherol was the most effective compound in reducing the reactive oxygen species (ROS) burden, both directly and indirectly. Adding 0.7 g/L of alpha-tocopherol led to an 18% improvement in biomass, from 6.29 g/L to 7.42 g/L. Moreover, the squalene concentration increased from 129.8 mg/L to 240.2 mg/L, indicating an 85% improvement, while the squalene yield increased by 63.2%, from 19.82 mg/g to 32.4 mg/g. Additionally, our comparative transcriptomics analysis suggested that several genes involved in glycolysis, pentose phosphate pathway, TCA cycle, and MVA pathway were overexpressed following alpha-tocopherol supplementation. The alpha-tocopherol supplementation also lowered ROS levels by binding directly to ROS generated in the fermentation medium and indirectly by stimulating genes that encode antioxidative enzymes, thereby decreasing the ROS burden. Our findings suggest that alpha-tocopherol supplementation can be an effective method for improving squalene production in Aurantiochytrium sp. TWZ-97 culture.

Saturated and Polyunsaturated Fatty Acids Production by Aurantiochytrium limacinum PKU#Mn4 on Enteromorpha Hydrolysate.

Thraustochytrids are unicellular marine heterotrophic protists, which have recently shown a promising ability to produce omega-3 fatty acids from lignocellulosic hydrolysates and wastewaters. Here we studied the biorefinery potential of the dilute acid-pretreated marine macroalgae (Enteromorpha) in comparison with glucose via fermentation using a previously isolated thraustochytrid strain (Aurantiochytrium limacinum PKU#Mn4). The total reducing sugars in the Enteromorpha hydrolysate accounted for 43.93% of the dry cell weight (DCW). The strain was capable of producing the highest DCW (4.32 ± 0.09 g/L) and total fatty acids (TFA) content (0.65 ± 0.03 g/L) in the medium containing 100 g/L of hydrolysate. The maximum TFA yields of 0.164 ± 0.160 g/g DCW and 0.196 ± 0.010 g/g DCW were achieved at 80 g/L of hydrolysate and 40 g/L of glucose in the fermentation medium, respectively. Compositional analysis of TFA revealed the production of equivalent fractions (% TFA) of saturated and polyunsaturated fatty acids in hydrolysate or glucose medium. Furthermore, the strain yielded a much higher fraction (2.61-3.22%) of eicosapentaenoic acid (C20:5n-3) in the hydrolysate medium than that (0.25-0.49%) in the glucose medium. Overall, our findings suggest that Enteromorpha hydrolysate can be a potential natural substrate in the fermentative production of high-value fatty acids by thraustochytrids.

Low dissolved oxygen supply functions as a global regulator of the growth and metabolism of Aurantiochytrium sp. PKU#Mn16 in the early stages of docosahexaenoic acid fermentation.

BACKGROUND: Thraustochytrids accumulate lipids with a high content of docosahexaenoic acid (DHA). Although their growth and DHA content are significantly affected by the dissolved oxygen (DO) supply, the role of DO on the transcriptional regulation of metabolism and accumulation of intracellular metabolites remains poorly understood. Here we investigate the effects of three different DO supply conditions (10%, 30%, and 50%) on the fed-batch culture of the Aurantiochytrium PKU#Mn16 strain to mainly reveal the differential gene expressions and metabolite profiles. RESULTS: While the supply of 10% DO significantly reduced the rates of biomass and DHA production in the early stages of fermentation, it achieved the highest amounts of biomass (56.7 g/L) and DHA (6.0 g/L) on prolonged fermentation. The transcriptome analyses of the early stage (24 h) of fermentation revealed several genes involved in the central carbon, amino acid, and fatty acid metabolism, which were significantly downregulated at a 10% DO level. The comparative metabolomics results revealed the accumulation of several long-chain fatty acids, amino acids, and other metabolites, supporting the transcriptional regulation under the influence of a low oxygen supply condition. In addition, certain genes involved in antioxidative systems were downregulated under 10% DO level, suggesting a lesser generation of reactive oxygen species that lead to oxidative damage and fatty acid oxidation. CONCLUSIONS: The findings of this study suggest that despite the slow growth and metabolism in the early stage of fermentation of Aurantiochytrium sp. PKU#Mn16, a constant supply of low dissolved oxygen can yield biomass and DHA content better than that with high oxygen supply conditions. The critical information gained in this study will help to further improve DHA production through bioprocess engineering strategies.

Subsurface Bacterioplankton Structure and Diversity in the Strongly-Stratified Water Columns within the Equatorial Eastern Indian Ocean.

The consequences of climate change may directly or indirectly impact the marine biosphere. Although ocean stratification has been recognized as one of the crucial consequences of ocean warming, its impacts on several critical aspects of marine microbes remain largely unknown in the Indian Ocean. Here, we investigate the effects of water stratification, in both surface and subsurface layers, on hydrogeographic parameters and bacterioplankton diversity within the equatorial eastern Indian Ocean (EIO). Strong stratification in the upper 200 m of equatorial EIO was detected with evidential low primary productivity. The vertical bacterioplankton diversity of the whole water columns displayed noticeable variation, with lower diversity occurring in the surface layer than in the subsurface layers. Horizontal heterogeneity of bacterioplankton communities was also in the well-mixed layer among different stations. SAR11 and Prochlorococcus displayed uncharacteristic low abundance in the surface water. Some amplicon sequence variants (ASVs) were identified as potential biomarkers for their specific depths in strongly-stratified water columns. Thus, barriers resulting from stratification are proposed to function as an 'ASV filter' to regulate the vertical bacterioplankton community diversity along the water columns. Overall, our results suggest that the effects of stratification on the structure and diversity of bacterioplankton can extend up to the bathypelagic zone in the strongly-stratified waters of the equatorial EIO. This study provides the first insight into the effect of stratification on the subsurface microbial communities in the equatorial eastern Indian Ocean.

Comparative Genomic Analyses of Cellulolytic Machinery Reveal Two Nutritional Strategies of Marine Labyrinthulomycetes Protists.

Labyrinthulomycetes are a group of ubiquitous and diverse unicellular Stramenopiles and have long been known for their vital role in ocean carbon cycling. However, their ecological function from the perspective of organic matter degradation remains poorly understood. This study reports high-quality genomes of two newly isolated Labyrinthulomycetes strains, namely, Botryochytrium sp. strain S-28 and Oblongichytrium sp. strain S-429, and provides molecular analysis of their ecological functions using comparative genomics and a biochemical assay. Our results suggest that Labyrinthulomycetes may occupy multiple ecological niches in marine ecosystems because of the significant differences in gene function among different genera. Certain strains could degrade wheat bran independently by secreting cellulase. The key glycoside hydrolase families (GH1, GH5, and GH9) related to cellulase and the functional domains of carbohydrate-active enzymes (CAZymes) were more enriched in their genomes. This group can actively participate in marine biochemical cycles as decomposers. In contrast, other strains that could not produce cellulase may thrive as "leftover scavengers" and act as a source of nutrients to the higher-trophic-level plankton. In addition, our findings emphasize the dual roles of endoglucanase, acting as both exo- and endoglucanases, in the process of cellulose degradation. Using genomic, biochemical, and phylogenetic analyses, our study provides a broader insight into the nutritional patterns and ecological functions of Labyrinthulomycetes. IMPORTANCE Unicellular heterotrophic eukaryotes are an important component of marine ecosystems. However, their ecological functions and modes of nutrition remain largely unknown. Our current understanding of marine microbial ecology is incomplete without integrating these heterotrophic microeukaryotes into the food web models. This study focuses on the unicellular fungus-like protists Labyrinthulomycetes and provides two high-quality genomes of cellulase-producing Labyrinthulomycetes. Our study uncovers the basis of their cellulase production by deciphering the results of genomic, biochemical, and phylogenetic analyses. This study instigates a further investigation of the molecular mechanism of organic matter utilization by Labyrinthulomycetes in the world's oceans.

Lignin-oxidizing and xylan-hydrolyzing Vibrio involved in the mineralization of plant detritus in the continental slope.

A large amount of terrigenous organic matter (TOM) is constantly transported to the deep sea. However, relatively little is known about the microbial mineralization of TOM therein. Our recent in situ enrichment experiments revealed that Vibrio is especially enriched as one of the predominant taxa in the cultures amended with natural plant materials in the deep sea. Yet their role in the mineralization of plant-derived TOM in the deep sea remains largely unknown. Here we isolated Vibrio strains representing dominant members of the enrichments and verified their potential to degrade lignin and xylan. The isolated strains were closely related to Vibrio harveyi, V. alginolyticus, V. diabolicus, and V. parahaemolyticus. Extracellular enzyme assays, and genome and transcriptome analyses revealed diverse peroxidases, including lignin peroxidase (LiP), catalase-peroxidase (KatG), and decolorizing peroxidase (DyP), which played an important role in the depolymerization and oxidation of lignin. Superoxide dismutase was found to likely promote lignin oxidation by supplying H2O2 to LiP, DyP, and KatG. Interestingly, these deep-sea Vibrio strains could oxidize lignin and hydrolyze xylan not only through aerobic pathway, but also through anaerobic pathway. Genome analysis revealed multiple anaerobic respiratory mechanisms, including the reductions of nitrate, arsenate, tetrathionate, and dimethyl sulfoxide. The strains showed the potential to anaerobically reduce sulfite and metal oxides of iron and manganese, in contrast the non-deep-sea Vibrio strains were not retrieved of genes involved in reduction of metal oxides. This is the first report about the lignin oxidation mechanisms in Vibrio and their role in TOM mineralization in anoxic and oxic environments of the marginal sea.

Nitrogen Starvation Enhances the Production of Saturated and Unsaturated Fatty Acids in Aurantiochytrium sp. PKU#SW8 by Regulating Key Biosynthetic Genes.

Nitrogen deprivation is known to improve lipid accumulation in microalgae and thraustochytrids. However, the patterns of fatty acid production and the molecular mechanisms underlying the accumulation of unsaturated and saturated fatty acids (SFAs) under nitrogen starvation remain largely unknown for thraustochytrids. In this study, batch culture experiments under nitrogen replete and nitrogen starvation conditions were performed, and the changes in the transcriptome of Aurantiochytrium sp. PKU#SW8 strain between these conditions were investigated. Our results showed improved yields of total fatty acids (TFAs), total unsaturated fatty acids, and total SFAs under nitrogen starvation, which suggested that nitrogen starvation favors the accumulation of both unsaturated and saturated fatty acids in PKU#SW8. However, nitrogen starvation resulted in a more than 2.36-fold increase of SFAs whereas a 1.7-fold increase of unsaturated fatty acids was observed, indicating a disproportionate increase in these groups of fatty acids. The fabD and enoyl-CoA hydratase genes were significantly upregulated under nitrogen starvation, supporting the observed increase in the yield of TFAs from 2.63 ± 0.22 g/L to 3.64 ± 0.16 g/L. Furthermore, the pfaB gene involved in the polyketide synthase (PKS) pathway was significantly upregulated under nitrogen starvation. This suggested that the increased expression of the pfaB gene under nitrogen starvation may be one of the explanations for the increased yield of docosahexaenoic acid by 1.58-fold. Overall, our study advances the current understanding of the molecular mechanisms that underlie the response of thraustochytrids to nitrogen deprivation and their fatty acid biosynthesis.

Optimal NaCl Medium Enhances Squalene Accumulation in Thraustochytrium sp. ATCC 26185 and Influences the Expression Levels of Key Metabolic Genes.

Squalene, a natural lipid of the terpenoid family, is well-recognized for its roles in regulating cholesterol metabolism, preventing tumor development, and improving immunity. For large-scale squalene production, the unicellular marine protists-thraustochytrids-have shown great potential. However, the growth of thraustochytrids is known to be affected by salt stress, which can eventually influence the squalene content. Here, we study the effects of an optimal concentration of NaCl on the squalene content and transcriptome of Thraustochytrium sp. ATCC 26185. Under the optimal culture conditions (glucose, 30 g/L; yeast extract, 2.5 g/L; and NaCl, 5 g/L; 28°C), the strain yielded 67.7 mg squalene/g cell dry weight, which was significantly greater than that (5.37 mg/g) under the unoptimized conditions. NaCl was determined as the most significant (R = 135.24) factor for squalene production among glucose, yeast extract, and NaCl. Further comparative transcriptomics between the ATCC 26185 culture with and without NaCl addition revealed that NaCl (5 g/L) influences the expression of certain key metabolic genes, namely, IDI, FAS-a, FAS-b, ALDH3, GS, and NDUFS4. The differential expression of these genes possibly influenced the acetyl-CoA and glutamate metabolism and resulted in an increased squalene production. Through the integration of bioprocess technology and transcriptomics, this report provides the first evidence of the possible mechanisms underscoring increased squalene production by NaCl.

Diversity, Abundance, and Ecological Roles of Planktonic Fungi in Marine Environments.

Fungi are considered terrestrial and oceans are a "fungal desert". However, with the considerable progress made over past decades, fungi have emerged as morphologically, phylogenetically, and functionally diverse components of the marine water column. Although their communities are influenced by a plethora of environmental factors, the most influential include salinity, temperature, nutrients, and dissolved oxygen, suggesting that fungi respond to local environmental gradients. The biomass carbon of planktonic fungi exhibits spatiotemporal dynamics and can reach up to 1 µg CL-1 of seawater, rivaling bacteria on some occasions, which suggests their active and important role in the water column. In the nutrient-rich coastal water column, there is increasing evidence for their contribution to biogeochemical cycling and food web dynamics on account of their saprotrophic, parasitic, hyper-parasitic, and pathogenic attributes. Conversely, relatively little is known about their function in the open-ocean water column. Interestingly, methodological advances in sequencing and omics approach, the standardization of sequence data analysis tools, and integration of data through network analyses are enhancing our current understanding of the ecological roles of these multifarious and enigmatic members of the marine water column. This review summarizes the current knowledge of the diversity and abundance of planktonic fungi in the world's oceans and provides an integrated and holistic view of their ecological roles.

Culturable Diversity of Thraustochytrids from Coastal Waters of Qingdao and Their Fatty Acids.

Thraustochytrids have gained significant attention in recent years because of their considerable ecological and biotechnological importance. Yet, the influence of seasons and habitats on their culturable diversity and lipid profile remains poorly described. In this study, a total of 58 thraustochytrid strains were isolated from the coastal waters of Qingdao, China. These strains were phylogenetically close to five thraustochytrid genera, namely Botryochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Sicyoidochytrium. Most of the isolated strains were classified into the genera Thraustochytrium and Oblongichytrium. Further diversity analysis revealed that samples collected from nutrient-rich habitats and during summer/fall yielded significantly higher culturable diversity of thraustochytrids than those from low-nutrient habitats and winter/spring. Moreover, sampling habitats and seasons significantly impacted the fatty acid profiles of the strains. Particularly, the Oblongichytrium sp. OC931 strain produced a significant amount (153.99 mg/L) of eicosapentaenoic acid (EPA), accounting for 9.12% of the total fatty acids, which was significantly higher than that of the previously reported Aurantiochytrium strains. Overall, the results of this study fill the gap in our current understanding of the culturable diversity of thraustochytrids in the coastal waters and the impact of the sampling habitats and seasons on their capacity for lipid accumulation.

Media Supplementation with Mannitol and Biotin Enhances Squalene Production of Thraustochytrium ATCC 26185 through Increased Glucose Uptake and Antioxidative Mechanisms.

Media supplementation with exogenous chemicals is known to stimulate the accumulation of important lipids produced by microalgae and thraustochytrids. However, the roles of exogenous chemicals in promoting and preserving the terpenoids pool of thraustochytrids have been rarely investigated. Here, we realized the effects of two media supplements-mannitol and biotin-on the biomass and squalene production by a thraustochytrid strain (Thraustochytrium sp. ATCC 26185) and elucidated their mechanism of action. A significant change in the biomass was not evident with the exogenous addition of these supplements. However, with mannitol (1 g/L) supplementation, the ATCC 26185 culture achieved the best concentration (642 ± 13.6 mg/L) and yield (72.9 ± 9.6 mg/g) of squalene, which were 1.5-fold that of the control culture (non-supplemented). Similarly, with biotin supplementation (0.15 mg/L), the culture showed 459 ± 2.9 g/L and 55.7 ± 3.2 mg/g of squalene concentration and yield, respectively. The glucose uptake rate at 24 h of fermentation increased markedly with mannitol (0.31 g/Lh-1) or biotin (0.26 g/Lh-1) supplemented culture compared with non-supplemented culture (0.09 g/Lh-1). In addition, the reactive oxygen species (ROS) level of culture supplemented with mannitol remained alleviated during the entire period of fermentation while it alleviated after 24 h with biotin supplementation. The ∆ROS with mannitol was better compared with biotin supplementation. The total antioxidant capacity (T-AOC) of the supplemented culture was more than 50% during the late stage (72-96 h) of fermentation. Our study provides the potential of mannitol and biotin to enhance squalene yield and the first lines of experimental evidence for their protective role against oxidative stress during the culture of thraustochytrids.

Chemical and Physical Culture Conditions Significantly Influence the Cell Mass and Docosahexaenoic Acid Content of Aurantiochytrium limacinum Strain PKU#SW8.

Thraustochytrids are well-known unicellular heterotrophic marine protists because of their promising ability to accumulate docosahexaenoic acid (DHA). However, the implications of their unique genomic and metabolic features on DHA production remain poorly understood. Here, the effects of chemical and physical culture conditions on the cell mass and DHA production were investigated for a unique thraustochytrid strain, PKU#SW8, isolated from the seawater of Pearl River Estuary. All the tested fermentation parameters showed a significant influence on the cell mass and concentration and yield of DHA. The addition of monosaccharides (fructose, mannose, glucose, or galactose) or glycerol to the culture medium yielded much higher cell mass and DHA concentrations than that of disaccharides and starch. Similarly, organic nitrogen sources (peptone, yeast extract, tryptone, and sodium glutamate) proved to be beneficial in achieving a higher cell mass and DHA concentration. PKU#SW8 was found to grow and accumulate a considerable amount of DHA over wide ranges of KH2PO4 (0.125-1.0 g/L), salinity (0-140% seawater), pH (3-9), temperature (16-36 °C), and agitation (140-230 rpm). With the optimal culture conditions (glycerol, 20 g/L; peptone, 2.5 g/L; 80% seawater; pH 4.0; 28 °C; and 200 rpm) determined based on the shake-flask experiments, the cell mass and concentration and yield of DHA were improved up to 7.5 ± 0.05 g/L, 2.14 ± 0.03 g/L, and 282.9 ± 3.0 mg/g, respectively, on a 5-L scale fermentation. This study provides valuable information about the fermentation conditions of the PKU#SW8 strain and its unique physiological features, which could be beneficial for strain development and large-scale DHA production.

Exogenous Antioxidants Improve the Accumulation of Saturated and Polyunsaturated Fatty Acids in Schizochytrium sp. PKU#Mn4.

Species of Schizochytrium are well known for their remarkable ability to produce lipids intracellularly. However, during their lipid accumulation, reactive oxygen species (ROS) are generated inevitably as byproducts, which if in excess results in lipid peroxidation. To alleviate such ROS-induced damage, seven different natural antioxidants (ascorbic acid, α-tocopherol, tea extract, melatonin, mannitol, sesamol, and butylated hydroxytoluene) were evaluated for their effects on the lipid accumulation in Schizochytrium sp. PKU#Mn4 using a fractional factorial design. Among the tested antioxidants, mannitol showed the best increment (44.98%) in total fatty acids concentration. However, the interaction effects of mannitol (1 g/L) and ascorbic acid (1 g/L) resulted in 2.26 ± 0.27 g/L and 1.45 ± 0.04 g/L of saturated and polyunsaturated fatty acids (SFA and PUFA), respectively, in batch fermentation. These concentrations were further increased to 7.68 ± 0.37 g/L (SFA) and 5.86 ± 0.03 g/L (PUFA) through fed-batch fermentation. Notably, the interaction effects yielded 103.7% and 49.6% increment in SFA and PUFA concentrations in batch fermentation. The possible mechanisms underlining those increments were an increased maximum growth rate of strain PKU#Mn4, alleviated ROS level, and the differential expression of lipid biosynthetic genes andupregulated catalase gene. This study provides an applicable strategy for improving the accumulation of SFA and PUFA in thraustochytrids by exogenous antioxidants and the underlying mechanisms.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation.

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Elemental Composition and Cell Mass Quantification of Cultured Thraustochytrids Unveil Their Large Contribution to Marine Carbon Pool.

The element stoichiometry of bacteria has received considerable attention because of their significant role in marine ecosystems. However, relatively little is known about the composition of major structural elements of the unicellular heterotrophic protists-thraustochytrids, despite their widely recognized contribution to marine nutrient cycling. Here, we analyze the cell volume and elemental C, N, H, and S cell content of seven cultured thraustochytrids, isolated from different marine habitats, in the exponential and stationary growth phases. We further derive the relationships between the cell volume and elemental C and N content of the cultured thraustochytrids. The cell volumes varied significantly (p < 0.001) among the isolates, with median values of 96.9 and 212.5 µm3 in the exponential and stationary phases, respectively. Our results showed a significantly higher percentage of C (64.0 to 67.5) and H (9.9 to 13.2) but a lower percentage of N (1.86 to 2.16) and S (0.34 to 0.91) in the stationary phase, along with marked variations of C and N fractions among isolates in the exponential phase. The cell C (5.7 to 203.7 pg) and N (0.65 to 6.1 pg) content exhibited a significant (p < 0.001) linear relationship with the cell volume (27.7 to 510 µm3). On further analysis of the relationship across the two growth phases, we found the equation (cell C (pg) = 0.356 × cell volume (µm3) + 20.922) for stationary phase cells more appropriate for C estimation of natural thraustochytrids. This study provides the first experimental evidence of higher cell C density than the current estimate and relatively larger C contribution of thraustochytrids than bacteria to the marine organic pool.

Disentangling the structure and function of mycoplankton communities in the context of marine environmental heterogeneity.

Mycoplankton are a diverse and ubiquitous component of marine environments with a suggested role in ocean biogeochemical cycling. Thus far, the patterns of their abundance, structure, and function against spatial environmental heterogeneity remains poorly understood. Based on in silico and experimental evaluation of multiple markers, we adopted the ITS1 region to determine the composition, guilds, and metabolic potential of mycoplankton communities in contrasting marine environments. The trophic status of estuarine (SB1 and SB2) and coastal (DB1 and DB2) sites, but not oceanic (OS) site, was the major factor that determined their abundances. While ascomycetous fungi dominated the estuarine and coastal sites, basidiomycetous fungi were found to dominate the oceanic site. The zoosporic fungi were relatively more abundant in SB1 and DB2 sites compared to the other sites. The relative abundances of the core fungi, namely Cystobasidium, Phlebia, Rhodotorula, Trichoderma, Alternaria, Penicillium, Malassezia, and Aspergillus varied widely across the sites. Additionally, several fungal genera unique to each site were also identified. DB2 site exhibited the lowest fungal richness while the OS site the highest. Conversely, the diversity and evenness were the lowest for the OS site but highest for the SB1 site. Temperature, pH, and chlorophyll-a were strongly associated with spatial diversity patterns. Of the 11 assigned guilds, some guilds particularly were not detected, including plant pathogen-wood saprotroph in DB2, the endophyte-plant pathogen in OS, the animal pathogen in SB1, and fungal parasite in DB1 and SB2. Within core functions-metabolism of amino acids, carbohydrates and energy, fatty acids and lipids, nitrogen, sulfur, and other compounds-several pathways showed spatial variations. Overall, this study not just broadens the taxonomic and metabolic repertoire of marine mycoplankton but also provides the first evidence of how these are shaped by site-scale environmental heterogeneity.

Different carbon and nitrogen sources regulated docosahexaenoic acid (DHA) production of Thraustochytriidae sp. PKU#SW8 through a fully functional polyunsaturated fatty acid (PUFA) synthase gene (pfaB).

Docosahexaenoic acid (DHA, C22:6) production in thraustochytrids is known to be mediated independently through polyunsaturated fatty acid (PUFA) synthase and fatty acid synthase systems. This study elucidates the unresolved effects of different carbon and nitrogen sources on the functionality of PUFA synthase subunit B (pfaB) and corresponding DHA production in Thraustochytriidae sp. PKU#SW8. Carbon and nitrogen sources showed significant effect on the pfaB gene expression and DHA production patterns, but these patterns did not correspond with each other, suggesting the strong role of substrates in differential induction of the two synthase systems. Nitrogen starvation increased DHA yield in parallel with upregulated gene expression, showing strong indication of PUFA synthase activity in N-deficient culture. The fully functional catalytic activity of PfaB subunit from strain PKU#SW8 in a heterologous host was also demonstrated. This study provides the direct evidence of pfaB gene actively for DHA biosynthesis in Thraustochytriidae sp. PKU#SW8.

Reactive oxygen species and their applications toward enhanced lipid accumulation in oleaginous microorganisms.

Oleaginous microorganisms are among the most promising alternative sources of lipids for oleochemicals and biofuels. However, in the course of lipid production, reactive oxygen species (ROS) are generated inevitably as byproducts of aerobic metabolisms. Although excessive accumulation of ROS leads to lipid peroxidation, DNA damage, and protein denaturation, ROS accumulation has been suggested to enhance lipid synthesis in these microorganisms. There are many unresolved questions concerning this dichotomous view of ROS influence on lipid accumulation. These include what level of ROS triggers lipid overproduction, what mechanisms and targets are vital and whether ROS act as toxic byproducts or cellular messengers in these microorganisms? Here we review the current state of knowledge on ROS generation, antioxidative defense system, the dual effects of ROS on microbial lipid production, and ROS-induced lipid peroxidation and accumulation mechanisms. Toward the end, the review summarizes strategies that enhance lipid production based on ROS manipulation.

Fed-batch fermentation of mixed carbon source significantly enhances the production of docosahexaenoic acid in Thraustochytriidae sp. PKU#Mn16 by differentially regulating fatty acids biosynthetic pathways.

This study reports comparative evaluation of the growth and DHA productivity of the thraustochytrid strain Thraustochytriidae PKU#Mn16 fermented with seven different substrate feeding strategies. Of these strategies, fed-batch fermentation of the mixed substrate (glucose & glycerol) yielded the maximum growth (52.2 ±â€¯1.5 g/L), DHA yield (Yp/s: 8.65) and productivity (100.7 ±â€¯2.9 mg/L-h), comparable with those of previously reported Aurantiochytrium strains. Transcriptomics analyses revealed that glucose upregulated some genes of the fatty acid synthase pathway whereas glycerol upregulated a few genes of the polyketide synthase pathway. Co-fermentation of the mixed substrate differentially regulated genes of these two pathways and significantly enhanced the DHA productivity. Furthermore, some genes involved in DNA replication, phagosome, carbon metabolism, and ß-oxidation were also found to alter significantly during the mixed-substrate fermentation. Overall, this study provides a unique strategy for enhancing growth and DHA productivity of the strain PKU#Mn16 and the first insight into the mechanisms underlying mixed-substrate fermentation.