Technologies for harvesting the microalgae for industrial applications: Current trends and perspectives.

Microalgae are emerging as a promising source for augmenting the supply of essential products to meet global demands in an environmentally sustainable manner. Despite the potential benefits of microalgae in industry, the high energy consumption for harvesting remains a significant obstacle. This review offers a comprehensive overview of microalgae harvesting technologies and their industrial applications, with particular emphasis on the latest advances in flocculation techniques. These cutting-edge methods have been applied to biodiesel production, food and nutraceutical processing, and wastewater treatment. Large-scale harvesting is still severely impeded by the high cost despite progress has been made in laboratory studies. In the future, cost-effective microalgal harvesting will rely on efficient resource utilization, including the use of waste materials and the reuse of media and flocculants. Additionally, precise regulation of biological metabolism will be necessary to overcome algal species-related limitations through the development of extracellular polymeric substance-induced flocculation technology.

Aggrephagy-related patterns in tumor microenvironment, prognosis, and immunotherapy for acute myeloid leukemia: a comprehensive single-cell RNA sequencing analysis.

Acute myeloid leukemia (AML) is a complex mixed entity composed of malignant tumor cells, immune cells and stromal cells, with intra-tumor and inter-tumor heterogeneity. Single-cell RNA sequencing enables a comprehensive study of the highly complex tumor microenvironment, which is conducive to exploring the evolutionary trajectory of tumor cells. Herein, we carried out comprehensive analyses of aggrephagy-related cell clusters based on single-cell sequencing for patients with acute myeloid leukemia. A total of 11 specific cell types (T, NK, CMP, Myeloid, GMP, MEP, Promono, Plasma, HSC, B, and Erythroid cells) using t-SNE dimension reduction analysis. Several aggrephagy-related genes were highly expressed in the 11 specific cell types. Using Monocle analysis and NMF clustering analysis, six aggrephagy-related CD8+ T clusters, six aggrephagy-related NK clusters, and six aggrephagy-related Mac clusters were identified. We also evaluated the ligand-receptor links and Cell-cell communication using CellChat package and CellChatDB database. Furthermore, the transcription factors (TFs) of aggrephagy-mediated cell clusters for AML were assessed through pySCENIC package. Prognostic analysis of the aggrephagy-related cell clusters based on R package revealed the differences in prognosis of aggrephagy-mediated cell clusters. Immunotherapy of the aggrephagy-related cell clusters was investigated using TIDE algorithm and public immunotherapy cohorts. Our study revealed the significance of aggrephagy-related patterns in tumor microenvironment, prognosis, and immunotherapy for AML.

Insights into carbon utilization under mixotrophic conditions in Chlamydomonas.

Mixotrophic microalgae cultivation with various carbon resources is considered as a strategy that could increase biomass. However, the mechanism of carbon utilization between inorganic carbon (IC) and organic carbon (OC) remains unknown. In this study, IC and OC consumption, chlorophyll fluorescence parameters, intracellular Nicotinamide adenine dinucleotide phosphate content and transcriptional changes in related genes were characterized. The results showed that IC was utilized preferentially, whereas 76% IC was consumed at 8 h. Subsequently, OC was the dominant carbon resource for fermentation. The cell density in the IC group was 100% higher than that in the group without IC at 24 h. Bicarbonate addition enhanced photosynthesis by dissipating less energy and generating more electrons and energy, which benefited OC assimilation. This finding was verified by qRT-PCR analysis. These results elucidate the carbon utilization mechanism under mixotrophic conditions, which provide clues for promoting microalgae growth by regulating carbon utilization.

Small peptide glutathione-induced bioflocculation for enhancing the food application potential of Chlorella pyrenoidosa.

Existing flocculants are used to enhance the harvesting efficiency of microalgae; however, harvesting biomass containing residues is unsuitable for food applications. In this study, a small peptide-induced bioflocculation technique was developed for harvesting microalgae, and the biomass was free of impurities. After seven days of cultivation with glutathione, 72 % flocculation efficiency of Chlorella pyrenoidosa was achieved after settling for 1 h. The nutrient composition of flocs depicted a higher protein (68.94 mg/L) and lipid (48.97 mg/L) content than those of the control (65.91 and 41.44 mg/L). The amino acid profiles of flocs showed the presence of more essential amino acids than in untreated cells. More omega polyunsaturated fatty acids, such as ω-3 and ω-9, accumulate in flocs. Extracellular polymeric substances, which induced bioflocculation, appeared markedly in flocs (150.02 mg/L) compared to the control (32.30 mg/L). This study provides novel insights into the residue-free algal harvesting method and obtained nutrition-enriched biomass.

Biosynthetic approaches to efficient assimilation of CO2 via photorespiration modification in plant chassis.

Plant chassis has emerged as the platform with great potential for bioproduction of high value-added products such as recombinant protein, vaccine and natural product. However, as the primary metabolic pathway, photorespiration results in the loss of photosynthetically fixed carbon compounds and limits the exploration of plant chassis. People are endeavored to reduce the photorespiration energy or carbon loss based on variation screening or genetic engineering. Insomuch as protein engineering of Rubisco has not resulted in the significant improvement of Rubisco specificity which is linked to the direct CO2 fixation, the biosynthetic approaches of photorespiration bypass are gaining much more attention and manifested great potentiality in conferring efficient assimilation of CO2 in plant chassis. In this review, we summarize the recent studies on the metabolic pathway design and implementation of photorespiration alternative pathway aiming to provide clues to efficiently enhance carbon fixation via the modification of photorespiration in plant chassis for bioproduction. These will benefit the development of plant synthetic metabolism for biorefineries via improvement of artificial carbon sequestration cycle, particularly for the mitigation of serious challenges such as extreme climate change, food and energy shortages in the future.

A Novel Salt-Bridge Electroflocculation Technology for Harvesting Microalgae.

Microalgae biomass, as a promising alternative feedstock, can be refined into biodiesel, pharmaceutical, and food productions. However, the harvesting process for quality biomass still remains a main bottleneck due to its high energy demand. In this study, a novel technique integrating alkali-induced flocculation and electrolysis, named salt-bridge electroflocculation (SBEF) with non-sacrificial carbon electrodes is developed to promote recovery efficiency and cost savings. The results show that the energy consumption decreased to 1.50 Wh/g biomass with a high harvesting efficiency of 90.4% under 300 mA in 45 min. The mean particle size of algae flocs increased 3.85-fold from 2.75 to 10.59 µm, which was convenient to the follow-up processing. Another major advantage of this method is that the salt-bridge firmly prevented cells being destroyed by the anode's oxidation and did not bring any external contaminants to algal biomass and flocculated medium, which conquered the technical defects in electro-flocculation. The proposed SBEF technology could be used as a low cost process for efficient microalgae harvest with high quality biomass.

Transcriptome analysis of lipid metabolism in response to cerium stress in the oleaginous microalga Nannochloropsis oculata.

Nannochloropsis oculata can accumulate large amounts of lipids under rare earth element (REE) conditions. However, the lipid accumulation mechanism responsible for REE stress has not been elucidated. In this study, the effects of cerium (the most abundant REE) on the growth and lipid accumulation of N. oculata were investigated. The de novo transcriptome data of N. oculata under cerium conditions were subsequently collected and analyzed. The results showed that N. oculata exhibited good cerium-resistance ability, showed slightly decrease in biomass but significantly increase in lipid content (55.8 % dry cell weight) under 6.0 mg/L cerium condition. Meanwhile, about 83.4 % cerium was biological fixated. Through transcriptome analysis, we found that the inhibited photosynthesis and carbon fixation pathways coupled with the stress-sensitive expression of ribosome biogenesis genes acclimatized the cells to REE stress. The active glycolysis pathway accelerated carbon flux to pyruvate and acetyl-CoA, and the upregulation of glycerol kinase and phosphatidate cytidylyltransferase genes further induced lipid accumulation. In addition, cerium downregulated the acyl-CoA oxidase and triacylglycerol lipase genes, which inhibited the degradation of lipids. Therefore, different responses to cerium demonstrate how N. oculata cells adapt to REE stress, and this knowledge may be used to extend our understanding of triacylglycerol (TAG) and the synthesis of other important metabolites.

Transcriptome analysis of Haematococcus pluvialis of multiple defensive systems against nitrogen starvation.

Haematococcus pluvialis could accumulate large amounts of triacylglycerol (TAG) and astaxanthin under various environmental stresses. To gain insights into the multiple defensive systems for carbon metabolism against nitrogen starvation, transcriptome analysis was performed. It was found that the genes related to carbon fixation, glycolysis, fatty acid and carotenoid biosynthesis pathways were up-regulated remarkably. Glyceraldehyde 3-phosphate (G3P) biosynthesis was accelerated with the enhanced C3 and C4 pathway. Meanwhile, the pyruvate kinase (PK) and pyruvate dehydrogenase E2 component (aceF) genes were significantly increased 12.9-fold and 13.9-fold, respectively, resulting more pyruvate and acetyl-CoA generation, which were beneficial to carotenoids and fatty acid biosynthesis. Methylerythritol 4-phosphate (MEP) pathway mediated carotenoid precursor isopentenyl diphosphate (IPP) synthesis, as the all eight related genes were up-regulated. The carbon flux toward astaxanthin biosynthesis with the increased astaxanthin pathway genes. The redistribution of carbon was also promoted for TAG accumulation. In addition, the up-regulation of diacylglycerol acyltransferase (DGAT) and phospholipid: diacylglycerol acyltransferase (PDAT) genes indicated that both acyl-CoA dependent and independent pathway regulated TAG accumulation. Therefore, this work reveals the multiple defensive mechanism for carbon metabolism in response to nitrogen starvation, which extended our understanding on the carotenoids, TAG and other important metabolites synthesis.

Enhancement of linoleic acid content stimulates astaxanthin esterification in Coelastrum sp.

Most natural astaxanthin is fatty acid-esterified in microalgae to prevent oxidation. However, the factors influencing astaxanthin esterification (AE) are poorly understood. In this study, obstacles to AE in Coelastrum sp. HA-1 were investigated. Only half of the astaxanthin molecules in HA-1 were esterified, but AE was stimulated with exogenous linoleic acid (LA) and ethanol treatment. Astaxanthin esters and total astaxanthin (TA) with exogenous LA were elevated to 3.82-fold and 2.18-fold of control levels, respectively. Treatment with 3% (v/v) ethanol enhanced transcription of the Δ12 fatty acid desaturase gene, which caused more oleic acid (OA) to be converted to LA. Furthermore, the contents of astaxanthin esters and TA were 2.42-fold and 1.61-fold control levels, respectively. These findings confirmed that AE was upregulated by increasing LA content. Thus, a large concentration of OA alone does not increase astaxanthin accumulation in HA-1, and a certain amount of LA was necessary for AE.

CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS.

Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae's strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a strain and revealed its high bicarbonate tolerant mechanism by analyzing carbonic anhydrase (CA). The strain was identified as Dunaliella salina HTBS with broad temperature adaptability (7-30°C). The strain grew well under 30% CO2 or 70gL(-1) NaHCO3. In comparison, two periplasm CAs (CAH1 and CAH2) were detected with immunoblotting analysis in HTBS but not in a non-HCO3(-)-tolerant strain. The finding was also verified by an enzyme inhibition assay in which only HTBS showed significant inhibition by extracellular CA inhibitor. Thus, we inferred that the extracellular CAH1 and CAH2 played a multifunctional role in the toleration of high bicarbonate by HTBS.

Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis.

[TREATMENT OF POSTEROLATERAL TIBIAL PLATEAU COLLAPSED AND SPLITED FRACTURES BY POSTEROMEDIAL AND ANTEROLATERAL APPROACHES].

OBJECTIVE: To explore the effectiveness of posteromedial and anterolateral approaches in the treatment of posterolateral tibial plateau collapsed and splited fractures. METHODS: Nineteen consecutive patients with posterolateral tibial plateau collapsed and splited fractures were treated between August 2010 and August 2013, and the clinical data were retrospectively analyzed. There were 13 males and 6 females, with an average age of 36.9 years (range, 25-75 years). All cases had closed fractures, involving 8 left sides and 11 right sides. Fractures involved posterior column according to the three-column classification based on CT scans; according to the Schatzker classification, all fractures were type II; according to the AO/Association for the Study of Internal Fixation classification (AO/OTA), all fractures were type 41-B3.1.2. The interval between injury and operation was 7-14 days (mean, 9 days). The reduction of collapsed fractures and implantation of artificial bone allograft were supported by T-shaped distal radius plate via the posteromedial approach. The splited fractures was fixed by less invasive stabilization system (LISS) plate via the anterolateral approach. RESULTS: The mean operation time was 69.0 minutes (range, 50-105 minutes). All incisions healed by first intention without neurovascular complications or wound infection. All patients were followed up 14-20 months (mean, 18.2 months). X-ray and CT examinations showed that collapsed tibial plateau and joint surface were completely corrected; bony union was obtained at 12 weeks on average (range, 10-16 weeks). No secondary collapsed fracture and knee varus or valgus occurred. The results were excellent in 12 cases, good in 5 cases, and fair in 2 cases with an excellent and good rate of 89.5% according to the Rasmussen's scoring system for knee function. CONCLUSION: The posteromedial approach combined with anterolateral approach for posterolateral tibial plateau fractures can fully expose the posterolateral aspects of the tibial plateau, and thus collapsed and splited fractures can be treated at the same time, which will lead to less operative time and good outcomes in the treatment of posterolateral tibial plateau collapsed and splited fractures.

Using ammonia for algae harvesting and as nutrient in subsequent cultures.

Microalgae have been considered as a promising feedstock for biofuels and greenhouse gas reduction. A low-cost harvesting technology without secondary contamination for down-stream extraction is a key requirement to make algal biofuel commercially viable. A novel harvesting method using ammonia as a flocculant to make the algal biomass settable was devised and studied. Another major advantage of this approach is that the ammonia added will be reused as fertilizer in the subsequent cultures. The results indicated that ammonia-induced flocculation led to more than 99% removal of algae at 12h. The OD(600) of algae growing in the ammonia-enriched flocculation medium treated with heating and CO(2) was 2 times than that of initial after 6 days. These results suggested that this flocculation method was efficient, convenient and allowed the reuse of the flocculated medium, therefore providing an option for economic harvesting and cultivation of microalgae.

Preparation and characteristics of bio-oil from the marine brown alga Sargassum patens C. Agardh.

The marine brown alga, Sargassum patens C. Agardh, floating on the Yellow Sea, was collected and converted to bio-oil through hydrothermal liquefaction with a modified reactor. A maximum yield of 32.1±0.2 wt.% bio-oil was obtained after 15 min at 340 °C, at a feedstock concentration of 15 g biomass/150 ml water, without using a catalyst. The bio-oil had a heating value of 27.1MJ/kg and contained water, lipid, alcohol, phenol, esters, ethers and aromatic compounds. The solid residue obtained had a high ash and oxygen content. The results suggest that S. patens C. Agardh has potential as biomass feedstock for fuel and chemical products.

[Association study on the mitochondrial genome region np16181-16193 variation with type 2 diabetes mellitus].

OBJECTIVE: To investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM). METHODS: Blood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms. RESULTS: The mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05). CONCLUSION: The mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.

[Mitochondrial D-Loop gene polymorphisms in the patients with type 2 diabetes mellitus].

Polymerase chain reaction (PCR) and direct nucleotide sequencing were used to analyze the mitochondrial D-loop gene of 199 Zhejiang patients with T2DM and 102 controls and the relationship between D-Loop gene variations and the main clinical symptoms. The mitochondrial D-Loop gene was a hypervariable area and np73A-G, np263A-G, np16223C-T, and np16519T-C were four high variations, and 29 unreported new variations were found. np193A-G, np234A-G, and np16108C-T were related to diabetes mellitus with family history. These results showed that there are various forms of polymorphism in mitochondrial DNA D-Loop gene in a Zhejiang population, some of which are related to diabetes mellitus.

Determination of oxymatrine and its metabolite matrine in rat blood and dermal microdialysates by high throughput liquid chromatography/tandem mass spectrometry.

Oxymatrine (OMT) and matrine (MT) are the major quinolizidine alkaloids found in certain Sophora plants, which have been extensively used in China for the treatment of viral hepatitis, cancer, cardiac diseases and skin diseases (such as atopic dermatitis and eczema). A precise, sensitive and high throughput LC-MS/MS was developed to determine OMT and its metabolite MT in rat blood and dermis collected using microdialysis technique. Microdialysis probes were inserted into the jugular vein/right atrium and dermis of Wistar rats, and 3% OMT gel (1g) was administered via topical application. The samples were collected and then injected into the LC-MS/MS system after adding the internal standard (codeine, CDN). Chromatographic separation was achieved in a run time of 2min on a reversed phase short-column (50mmx2.1mm, 3.5microm). The mobile phase for column separation was methanol-ammonium formate (pH 5.0; 25mM) (70:30, v/v) with a flow rate of 0.3mL/min. A diverter valve was installed post-LC column for desalting. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for OMT, MT and IS was m/z 265.0-->247.3, 249.1-->148.3 and 300.0-->215.2, respectively. The lower limit of quantification (LLOQ) for OMT and MT was 0.5ng/mL. The calibration curves were linear over the range of 0.5-1000ng/mL for OMT and MT with a coefficient of determination >0.999. This selective and sensitive method is useful for the determination of OMT and MT and in the pharmacokinetic studies of these compounds. The blood and dermal concentration-time profile of OMT and its metabolite MT suggest that the limiting factor for dermal metabolism is the low capacity of enzymes in the skin rather than the quantity of penetrated OMT.

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification.

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy-Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.

Protective effect of nitric oxide on iron deficiency-induced oxidative stress in maize (Zea mays).

The effects of nitric oxide (NO) in protecting maize (Zea mays) leaves against iron deficiency-induced oxidative stress were investigated. The increased contents of hydrogen peroxide (H(2)O(2)) and superoxide (O(2)(-)*) due to iron deficiency suggested oxidative stress. The increased contents of thiobarbituric acid-reacting substances (TBARS) and the decreased contents of protein-bound thiol (PT) and non-protein-bound thiol (NPT) indicated iron deficiency-induced oxidative damage on proteins and lipids. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, partially reversed iron deficiency-induced retardation of plant growth as well as chlorosis. Reduced contents of H(2)O(2), O(2)(-)*, TBARS and increased contents of PT and NPT also indicated that NO alleviated iron deficiency-induced oxidative damage. The activities of SOD and GR decreased sharply while the activities of CAT, POD and APX increased under SNP treatment. Our data suggest that NO can protect maize plants from iron deficiency-induced oxidative stress by reacting with ROS directly or by changing activities of ROS-scavenging enzymes.