Bioprocess conditions and regulation factors to optimize squalene production in thraustochytrids.

Squalene is a widely distributed natural triterpene, as it is a key precursor in the biosynthesis of all sterols. It is a compound of high commercial value worldwide because it has nutritional, medicinal, pharmaceutical, and cosmetic applications, due to its different biological properties. The main source of extraction has been shark liver oil, which is currently unviable on a larger scale due to the impacts of overexploitation. Secondary sources are mainly vegetable oils, although a limited one, as they allow low productive yields. Due to the diversity of applications that squalene presents and its growing demand, there is an increasing interest in identifying sustainable sources of extraction. Wild species of thraustochytrids, which are heterotrophic protists, have been identified to have the highest squalene content compared to bacteria, yeasts, microalgae, and vegetable sources. Several studies have been carried out to identify the bioprocess conditions and regulation factors, such as the use of eustressors that promote an increase in the production of this triterpene; however, studies focused on optimizing their productive yields are still in its infancy. This review includes the current trends that also comprises the advances in genetic regulations in these microorganisms, with a view to identify the culture conditions that have been favorable in increasing the production of squalene, and the influences that both bioprocess conditions and applied regulation factors partake at a metabolic level.

Effects of sodium selenite on the growth, biochemical composition and selenium biotransformation of the filamentous microalga Tribonema minus.

This study aimed to investigate the physiological and biochemical responses of filamentous microalga Tribonema minus to different Na2SeO3 concentrations and its selenium absorption and metabolism to evaluate the potential in treating selenium-containing wastewater. The results showed that low Na2SeO3 concentrations promoted growth by increasing chlorophyll content and antioxidant capacity, whereas high concentrations caused oxidative damage. Although Na2SeO3 exposure reduced lipid accumulation compared with the control, it significantly increased carbohydrate, soluble sugar, and protein contents, with the highest carbohydrate productivity of 117.97 mg/L/d at 0.5 mg/L Na2SeO3. Furthermore, this alga effectively absorbed Na2SeO3 in the growth medium and converted most of it into volatile selenium and a small part into organic selenium (predominantly as selenocysteine), showing strong selenite removal efficacy. This is the first report on the potential of T. minus to produce valuable biomass while removing selenite, providing new insights into the economic feasibility of bioremediation of selenium-containing wastewater.

Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica with various nitrogen/phosphorus nutrients for CO2 fixation.

To determine the optimal CO2 concentration for microalgal biomass cultivated with industrial flue gas and improve carbon fixation capacity and biomass production. Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica (N. oceanica) with various nitrogen/phosphorus (N/P) nutrients for CO2 fixation were comprehensively clarified. At 100 % N/P nutrients, the optimum CO2 concentration was 70 % and the maximum biomass production of microalgae was 1.57 g/L. The optimum CO2 concentration was 50 % for N or P deficiency and 30 % for both N and P deficiency. The optimal combination of CO2 concentration and N/P nutrients caused significant up regulation of proteins related to photosynthesis and cellular respiration in the microalgae, enhancing photosynthetic electron transfer efficiency and carbon metabolism. Microalgal cells with P deficiency and optimal CO2 concentration expressed many phosphate transporter proteins to enhance P metabolism and N metabolism to maintain a high carbon fixation capacity. However, inappropriate combination of N/P nutrients and CO2 concentrations caused more errors in DNA replication and protein synthesis, generating more lysosomes and phagosomes. This inhibited carbon fixation and biomass production in the microalgae with increased cell apoptosis.

Enhancement of Squalene Production by Constitutive Expression of the 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Aurantiochytrium sp. 18W-13a.

Squalene has a wide range of applications in the industry sectors of dietary supplements, cosmetics, immunization, and pharmaceuticals. Yet, suitable organisms as the source of squalene are limited. It is reported that the thraustochytrid Aurantiochytrium sp. strain 18W-13a can accumulate high content of squalene. However, squalene production in this organism is fluctuated under various conditions and is not yet optimized for commercialization. In this organism, the mevalonate pathway supplies isopentenyl pyrophosphate, the initial substrate for squalene production. In this pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzyme. We found that the HMGR activity had a strong positive correlation with the squalene contents in the strain. We constitutively expressed the HMGR in this organism and found that the transformant showed increased and stable production of squalene as well as carotenoids and biomass. These results clearly indicated that the HMGR expression is the bottleneck of squalene synthesis in Aurantiochytrium sp.

Osteogenic activities of four calcium-chelating microalgae peptides.

BACKGROUND: Adequate calcium intake is necessary to prevent osteoporosis, which poses significant public health challenges. The natural bioactive peptide calcium chelates have been regarded as superior calcium supplements. Microalgae peptides are regarded as potential candidates for protection from bone loss in osteoporosis. This study aimed to prepare microalgae calcium-chelating peptides from four microalgae proteins and assess their osteogenic activities in osteoporosis-like zebrafish. RESULTS: After in vitro gastrointestinal digestion, 4.42% Chlorella pyrenoidosa protein, 2.74% Nannochloropsis oceanica protein, 6.07% Arthospira platensis protein and 10.47% Dunaliella salina protein were retained. The calcium-chelating capacities of four microalgae protein hydrolysates (MPHs) ranged from 14.10 ± 7.16% to 34.11 ± 9.34%. CaCl2 addition increased the maximum absorption peaks, absorption intensities and particle sizes of MPHs. Calcium-chelating MPHs showed stronger osteogenic activities than MPHs in the osteoporosis-like zebrafish model, with significantly increased mineralized tissue area and integrated optical density. CONCLUSION: Microalgae proteins have favorable digestibilities. Among the four MPHs, Nannochloropsis oceanica protein hydrolysates showed the highest calcium-chelating capacity, which might be due to its high degree of hydrolysis after in vitro digestion and high content of Ser, Tyr, Thr, Asp and Glu. The absorption intensities and particle sizes of MPHs both increased after calcium addition. MPH treatment could reverse dexamethasone-induced osteoporosis of zebrafish, and MPHs-Ca chelates showed higher osteogenic activities in osteoporosis-like phenotype zebrafish. © 2022 Society of Chemical Industry.

Xanthophylls from the Sea: Algae as Source of Bioactive Carotenoids.

Algae are considered pigment-producing organisms. The function of these compounds in algae is to carry out photosynthesis. They have a great variety of pigments, which can be classified into three large groups: chlorophylls, carotenoids, and phycobilins. Within the carotenoids are xanthophylls. Xanthophylls (fucoxanthin, astaxanthin, lutein, zeaxanthin, and ß-cryptoxanthin) are a type of carotenoids with anti-tumor and anti-inflammatory activities, due to their chemical structure rich in double bonds that provides them with antioxidant properties. In this context, xanthophylls can protect other molecules from oxidative stress by turning off singlet oxygen damage through various mechanisms. Based on clinical studies, this review shows the available information concerning the bioactivity and biological effects of the main xanthophylls present in algae. In addition, the algae with the highest production rate of the different compounds of interest were studied. It was observed that fucoxanthin is obtained mainly from the brown seaweeds Laminaria japonica, Undaria pinnatifida, Hizikia fusiformis, Sargassum spp., and Fucus spp. The main sources of astaxanthin are the microalgae Haematococcus pluvialis, Chlorella zofingiensis, and Chlorococcum sp. Lutein and zeaxanthin are mainly found in algal species such as Scenedesmus spp., Chlorella spp., Rhodophyta spp., or Spirulina spp. However, the extraction and purification processes of xanthophylls from algae need to be standardized to facilitate their commercialization. Finally, we assessed factors that determine the bioavailability and bioaccesibility of these molecules. We also suggested techniques that increase xanthophyll's bioavailability.

Novel Insights into Phosphorus Deprivation Boosted Lipid Synthesis in the Marine Alga Nannochloropsis oceanica without Compromising Biomass Production.

Nannochloropsis oceanica represents a preferred oleaginous alga for producing lipids. Here we found that phosphorus deprivation (PD) caused a severe decrease in protein and a considerable increase in lipids including triacylglycerol (TAG), yet it had little effect on the carbohydrate level and biomass production of N. oceanica. The combinatorial analysis by integrating physiological, biochemical, and transcriptomic data unraveled the molecular mechanisms underlying PD-induced lipid accumulation. Albeit attenuating the Calvin-Benson cycle, PD stimulated the C4-like pathway to maintain CO2 fixation for biomass production. PD attenuated nitrogen utilization and enhanced protein catabolism thus leading to protein decrease, from which the carbon was likely salvaged into the stimulated tricarboxylic acid cycle for supplying lipid synthesis with carbon precursors. The impairment of TAG catabolism by downregulating certain lipases rather than the stimulation of TAG assembly pathways contributed to PD-boosted TAG increase. These findings provide novel insights into PD-induced lipogenesis without compromising biomass production by N. oceanica.

Co-production of DHA and squalene by thraustochytrid from forest biomass.

Omega-3 fatty acids, and specifically docosahexaenoic acid (DHA), are important and essential nutrients for human health. Thraustochytrids are recognised as commercial strains for nutraceuticals production, they are group of marine oleaginous microorganisms capable of co-synthesis of DHA and other valuable carotenoids in their cellular compartment. The present study sought to optimize DHA and squalene production by the thraustochytrid Schizochytrium limacinum SR21. The highest biomass yield (0.46 g/gsubstrate) and lipid productivity (0.239 g/gsubstrate) were observed with 60 g/L of glucose, following cultivation in a bioreactor, with the DHA content to be 67.76% w/wtotal lipids. To reduce costs, cheaper feedstocks and simultaneous production of various value-added products for pharmaceutical or energy use should be attempted. To this end, we replaced pure glucose with organosolv-pretreated spruce hydrolysate and assessed the simultaneous production of DHA and squalene from S. limacinum SR21. After the 72 h of cultivation period in bioreactor, the maximum DHA content was observed to 66.72% w/wtotal lipids that was corresponded to 10.15 g/L of DHA concentration. While the highest DHA productivity was 3.38 ± 0.27 g/L/d and squalene reached a total of 933.72 ± 6.53 mg/L (16.34 ± 1.81 mg/gCDW). In summary, we show that the co-production of DHA and squalene makes S. limacinum SR21 appropriate strain for commercial-scale production of nutraceuticals.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction.

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L-1. Increasing the nutrient level in the medium by supplementation of 9 g L-1 KH2PO4 and 20 g L-1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L-1, respectively) were achieved when 35 g L-1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Supplemental Docosahexaenoic-Acid-Enriched Microalgae Affected Fatty Acid and Metabolic Profiles and Related Gene Expression in Several Tissues of Broiler Chicks.

This experiment was to enrich docosahexaenoic acid (DHA) in broiler tissues through feeding a DHA-rich microalgal biomass and to explore the underlying metabolic and molecular mechanisms. Hatchling Cornish male broilers (total = 192) were fed a corn-soybean meal basal diet containing a full-fatted microalgae ( Aurantiochytrium) at 0%, 1%, 2%, and 4% for 6 weeks ( n = 6 cages/treatment, 8 birds/cage). The inclusion of microalgae led to dose-dependent ( P < 0.01) enrichments of DHA and decreases ( P < 0.01) of n-6/n-3 fatty acids (FAs) in plasma, liver, muscle, and adipose tissue. The microalgae supplementation also lowered ( P < 0.05-0.1) nonesterified FAs concentrations in the plasma, liver and adipose tissue. The mRNA abundances of most assayed genes involved in lipid metabolism were decreased ( P < 0.05) in the liver but elevated ( P < 0.05) in the adipose in response to the biomass supplementation. In conclusion, the biomass-resultant DHA enrichments in the broiler tissues were associated with a distinctive difference in the expression of lipid metabolism-controlling genes between the liver and adipose tissue.

Development of a closed-loop process for fusel alcohol production and nutrient recycling from microalgae biomass.

Improving the economic feasibility is necessary for algae-based processes to achieve commercial scales for biofuels and bioproducts production. A closed-loop system for fusel alcohol production from microalgae biomass with integrated nutrient recycling was developed, which enables the reuse of nitrogen and phosphorus for downstream application and thus reduces the operational requirement for external major nutrients. Mixed fusel alcohols, primarily isobutanol and isopentanol were produced from Microchloropsis salina hydrolysates by an engineered E. coli co-culture. During the process, cellular nitrogen from microalgae biomass was converted into ammonium, whereas cellular phosphorus was liberated by an osmotic shock treatment. The formation of struvite from the liberated ammonium and phosphate, and the subsequent utilization of struvite to support M. salina cultivation was demonstrated. The closed loop system established here should help overcome one of the identified economic barriers to scale-up of microalgae production, and enhance the sustainability of microalgae-based chemical commodities production.

Different DHA or EPA production responses to nutrient stress in the marine microalga Tisochrysis lutea and the freshwater microalga Monodus subterraneus.

This study investigated the effect of nitrogen (N) and phosphorous (P) stress on the production of DHA or EPA and total fatty acids (TFAs) in the marine microalga Tisochrysis lutea and the freshwater microalga Monodus subterraneus. Five N or P starvation/limitation conditions (N sufficient and P limited, N sufficient and P starved, N starved and P sufficient, N starved and P limited, and N and P starved) and one N and P sufficient condition (control) were studied. The results demonstrated that the proportion of DHA or EPA among TFAs and production in the microalgae suspensions decreased (57%, 73% for N stress and 18%, 51% for P stress, respectively) under N or P stress in both microalgae compared with the N and P sufficient group. Differently, DHA dry weight content of T. lutea decreased significantly, and EPA dry weight content of M. subterraneus decreased slightly under N starved conditions. Clear differences in TFA content/production and the relationship between TFA and DHA or EPA production/content and CO2 fixation were observed between the two microalgae. These results give a new sight on the difference between marine microalgae and freshwater microalgae. Meanwhile, it gave a potential application to produce DHA or EPA and TFA combining with CO2 fixation by these microalgae.

Sunflower Oil and Nannochloropsis oculata Microalgae as Sources of Unsaturated Fatty Acids for Mitigation of Methane Production and Enhancing Diets' Nutritive Value.

The objective of this assay was to investigate the effect of adding sunflower oil, Nannochloropsis oculata microalgae and their mixture at 0, 1, 2, 3, 4, and 5% to three total mixed rations (TMRs) with different concentrate:forage ratios (40C:60F, 50C:50F, and 60C:40F) on in vitro gas production (GP), methane (CH4) production, and nutrient degradability. Asymptotic GP, GP rate, CH4 concentration/g acid detergent fiber (ADF), dry matter (DM) degradability (DMD), short chain fatty acids (SCFAs), and ruminal bacteria population increased, but neutral detergent fiber (NDF) degradability (NDFD), ADF degradability (ADFD), and protozoa count decreased with increasing concentrate level in the TMR. Methane production/g DM and NDF was higher for 50C:50F TMR. Sunflower oil reduced asymptotic GP, lag time, CH4 production/g ADF, ammonia-N (NH3-N), and SCFA. Compared to the control treatments, additives decreased GP rate, while sunflower oil/N. oculata mixture increased DMD and NDFD. All additives at 5% increased GP rate and lag time and decreased CH4 production/g DM, ADF, and NDF, ruminal NH3-N, and protozoa count. All additives at 2% increased DMD, NDFD and ADFD, SCFA, and bacteria population. Supplementation of TMR, containing different concentrate:forage ratios, with sunflower oil, N. oculata, and sunflower oil/N. oculata mixture at different doses modified in vitro GP, CH4 production, and nutrient degradability.

Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening.

Schizochytrium sp. accumulates valuable polyunsaturated fatty acid (PUFA), such as docosahexaenoic acid (DHA). In order to increase DHA synthesis in this microorganism, physical or chemical mutagenesis aided with powerful screening methods are still preferable, as its DHA synthetic pathway has not yet been clearly defined for gene manipulation. To breed this agglomerate microorganism of thick cell wall and rather large genome for increasing lipid content and DHA percentage, a novel strategy of atmospheric and room temperature plasma (ARTP) mutagenesis coupled with stepped malonic acid (MA) and zeocin resistance screening was developed. The final resulted mutant strain mz-17 was selected with 1.8-fold increased DHA production. Accompanied with supplementation of Fe2+ in shake flask cultivation, DHA production of 14.0 g/L on average was achieved. This work suggests that ARTP mutation combined with stepped MA and zeocin resistance screening is an efficient method of breeding Schizochytrium sp. of high DHA production, and might be applied on other microorganisms for obtaining higher desired PUFA products.

Isolation and molecular characterization of Thraustochytrium strain isolated from Antarctic Peninsula and its biotechnological potential in the production of fatty acids

ABSTRACT Thraustochytrids are unicellular protists belonging to the Labyrinthulomycetes class, which are characterized by the presence of a high lipid content that could replace conventional fatty acids. They show a wide geographic distribution, however their diversity in the Antarctic Region is rather scarce. The analysis based on the complete sequence of 18S rRNA gene showed that strain 34-2 belongs to the species Thraustochytrium kinnei, with 99% identity. The total lipid profile shows a wide range of saturated fatty acids with abundance of palmitic acid (16:0), showing a range of 16.1-19.7%. On the other hand, long-chain polyunsaturated fatty acids, mainly docosahexaenoic acid and eicosapentaenoic acid are present in a range of 24-48% and 6.1-9.3%, respectively. All factors analyzed in cells (biomass, carbon consumption and lipid content) changed with variations of culture temperature (10 °C and 25 °C). The growth in glucose at a temperature of 10 °C presented the most favorable conditions to produce omega-3fatty acid. This research provides the identification and characterization of a Thraustochytrids strain, with a total lipid content that presents potential applications in the production of nutritional supplements and as well biofuels.

Isolation and molecular characterization of Thraustochytrium strain isolated from Antarctic Peninsula and its biotechnological potential in the production of fatty acids.

Thraustochytrids are unicellular protists belonging to the Labyrinthulomycetes class, which are characterized by the presence of a high lipid content that could replace conventional fatty acids. They show a wide geographic distribution, however their diversity in the Antarctic Region is rather scarce. The analysis based on the complete sequence of 18S rRNA gene showed that strain 34-2 belongs to the species Thraustochytrium kinnei, with 99% identity. The total lipid profile shows a wide range of saturated fatty acids with abundance of palmitic acid (16:0), showing a range of 16.1-19.7%. On the other hand, long-chain polyunsaturated fatty acids, mainly docosahexaenoic acid and eicosapentaenoic acid are present in a range of 24-48% and 6.1-9.3%, respectively. All factors analyzed in cells (biomass, carbon consumption and lipid content) changed with variations of culture temperature (10°C and 25°C). The growth in glucose at a temperature of 10°C presented the most favorable conditions to produce omega-3fatty acid. This research provides the identification and characterization of a Thraustochytrids strain, with a total lipid content that presents potential applications in the production of nutritional supplements and as well biofuels.

Long-duration effect of multi-factor stresses on the cellular biochemistry, oil-yielding performance and morphology of Nannochloropsis oculata.

Microalga Nannochloropsis oculata is a promising alternative feedstock for biodiesel. Elevating its oil-yielding capacity is conducive to cost-saving biodiesel production. However, the regulatory processes of multi-factor collaborative stresses (MFCS) on the oil-yielding performance of N. oculata are unclear. The duration effects of MFCS (high irradiation, nitrogen deficiency and elevated iron supplementation) on N. oculata were investigated in an 18-d batch culture. Despite the reduction in cell division, the biomass concentration increased, resulting from the large accumulation of the carbon/energy-reservoir. However, different storage forms were found in different cellular storage compounds, and both the protein content and pigment composition swiftly and drastically changed. The analysis of four biodiesel properties using pertinent empirical equations indicated their progressive effective improvement in lipid classes and fatty acid composition. The variation curve of neutral lipid productivity was monitored with fluorescent Nile red and was closely correlated to the results from conventional methods. In addition, a series of changes in the organelles (e.g., chloroplast, lipid body and vacuole) and cell shape, dependent on the stress duration, were observed by TEM and LSCM. These changes presumably played an important role in the acclimation of N. oculata to MFCS and accordingly improved its oil-yielding performance.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 µg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Nutrient recycle from defatted microalgae (Aurantiochytrium) with hydrothermal treatment for microalgae cultivation.

Defatted heterotrophic microalgae (Aurantiochytrium limacinum SR21) was treated with high temperature water (175-350°C, 10-90min) to obtain nitrogen and phosphorous nutrients as a water soluble fraction (WS). Yields of nitrogen and phosphorous recovered in WS varied from 38 to 100% and from 57 to 99%, respectively. Maximum yields of nitrogen containing compounds in WS were proteins (43%), amino acids (12%) and ammonia (60%) at treatment temperatures of 175, 250 and 350°C, respectively. Maximum yield of phosphorous in WS was 99% at a treatment temperature of 250°C. Cultivation experiments of microalgae (A. limacinum SR21) using WS obtained at 200 and 250°C showed positive growth. Water soluble fractions from hydrothermal treatment of defatted microalgae are effective nitrogen and phosphorous nutrient sources for microalgae cultivation.

Metagenomic Analysis of Genes Encoding Nutrient Cycling Pathways in the Microbiota of Deep-Sea and Shallow-Water Sponges.

Sponges host complex symbiotic communities, but to date, the whole picture of the metabolic potential of sponge microbiota remains unclear, particularly the difference between the shallow-water and deep-sea sponge holobionts. In this study, two completely different sponges, shallow-water sponge Theonella swinhoei from the South China Sea and deep-sea sponge Neamphius huxleyi from the Indian Ocean, were selected to compare their whole symbiotic communities and metabolic potential, particularly in element transformation. Phylogenetically diverse bacteria, archaea, fungi, and algae were detected in both shallow-water sponge T. swinhoei and deep-sea sponge N. huxleyi, and different microbial community structures were indicated between these two sponges. Metagenome-based gene abundance analysis indicated that, though the two sponge microbiota have similar core functions, they showed different potential strategies in detailed metabolic processes, e.g., in the transformation and utilization of carbon, nitrogen, phosphorus, and sulfur by corresponding microbial symbionts. This study provides insight into the putative metabolic potentials of the microbiota associated with the shallow-water and deep-sea sponges at the whole community level, extending our knowledge of the sponge microbiota's functions, the association of sponge- microbes, as well as the adaption of sponge microbiota to the marine environment.