A Novel Hybrid Model Based on Machine and Deep Learning Techniques for the Classification of Microalgae

Classification and monitoring of microalgae species in aquatic ecosystems are important for understanding population dynamics.However,manual classification of algae is a time-consuming method and requires a lot of effort with expertise due to the large number of families and genera in its classification.The recognition of microalgae species has become an increasingly important research area in image recognition in recent years.In this study,machine learning and deep learning methods were proposed to classify images of 12 different microalgae species in order to successfully classify algae cells.8 Different novel models(MobileNetV3Small-Lr,MobileNetV3Small-Rf,MobileNetV3Small-Xg,MobileNetV3Large-Lr,MobileNetV3Large-Rf,MobileNetV3Large-Xg,Mobile-NetV3Small-Improved and MobileNetV3Large-Improved)have been proposed to classify these microalgae species.Among these proposed model structures,the best classification accuracy rate was 92.22%and the loss rate was 0.72,obtained from the MobileNetV3Large-Improved model structure.In addition,as a result of the experimental results obtained,metrics such as the confusion matrix,which can meet the experts in the correct diagnosis of microalgae species,were also evaluated.This research may in the future open a new avenue for the development of a cost-effective,highly sensitive computer-based system for the use of image analysis and deep learning techniques for the identification and classification of different microalgae.

Experimental Study of Microalgae Cultivation under Selective Illumination by Ag/CoSO_(4)for Bioelectrode Materials Preparation

Microalgae biomass is an ideal precursor to prepare renewable carbon materials,which has broad application.The bioaccumulation efficiency(lipids,proteins,carbohydrates)and biomass productivity of microalgae are influenced by spectroscopy during the culture process.In this study,a bilayer plate-type photobioreactor was designed to cultivate Chlorella protothecoides with spectral selectivity by nanofluids.Compared to culture without spectral selectivity,the spectral selectivity of Ag/CoSO_(4)nanofluids increased microalgae biomass by 5.76%,and the spectral selectivity of CoSO_(4)solution increased by 17.14%.In addition,the spectral selectivity of Ag/CoSO_(4)nanofluids was more conducive to the accumulation of nutrients(29.46%lipids,50.66%proteins,and 17.86%carbohydrates)in microalgae.Further cultured chlorella was utilized to prepare bioelectrode materials,it was found that algal based biochar had a good pore structure(micro specific surface area:1627.5314 m^(2)/g,average pore size:0.21294 nm).As the current density was 1 A/g,the specific capacitance reached 230 F/g,appearing good electrochemical performance.

Impacts of Sulfur and Microalgae Co-fertilization on Saline-alkaline Soil of Sunflower Field in Hetao Irrigation District

[Objectives]To evaluate the impacts of the elemental sulfur(S 0)and micro-algae(MA)co-fertilization on saline-alkaline soil of sunflower field in the Hetao Irrigation District(HID).[Methods]The greenhouse pot experiment was conducted with four treatments:control(CK),single S 0 fertilization(S),single MA fertilization(A),and S 0 and MA co-fertilization(SA)for comparing the selected soil properties and sunflower plant heights and weights in different treatments.[Results]The results showed that the mean soil organic matter(SOM)under the SA(25.08 g/kg)was significantly higher than that for the CK(20.59 g/kg),S(22.47 g/kg),and A(22.95 g/kg).The mean pH under the SA(7.75)was significantly lower than that for the CK(8.14),S(7.82),and A(7.96).The mean soil exchangeable Na+concentration under the SA was significantly lower than that for the S.The mean soil electrical conductivity(EC)under the SA was 9.76%lower than that for the S.The means of Cl-(1.22 g/kg)and SO 2-4(1.90 g/kg)in soil under the SA were lower than that for the S(1.30,2.06 g/kg)and A(1.31,1.97 g/kg),respectively.For plant height 3 at the late stage of plant growth,the mean plant height 3 under the SA(89.00 cm)was higher than that of the CK(69.60 cm)and A(74.33 cm).The total weights of the fresh sunflower heads,fresh stems,and dry seeds under the SA were higher than that for the CK,S,and A.[Conclusions]In conclusion,the S 0 and MA co-fertilization had positive effects on improving saline-alkaline soils,the soil under the S 0 and MA co-fertilization could be better conditions for promoting sunflower growth than that for the S,Z,and CK,and thereby the S 0 and MA co-fertilization could be a new idea to improve saline-alkaline soil in the cold and arid regions.

Heavy metal pollution of river water and eco-friendly remediation using potent microalgal species

Pollution of rivers is mainly caused by anthropogenic activities such as discharge of effluent from industrial facilities,maintenance of sewage/effluent treatment plants,and dumping of solid waste on river banks.This study dealt with the pollution issues of the Cooum River in the well-known city of Chennai in South India.Water samples from 27 locations were collected and analyzed for 12 elements,including Ba,B,and Al,as well as heavy metals such as Pb,Cr,Mn,Fe,Co,Ni,Cu,Zn,and Cd.The samples showed levels of these elements that exceeded World Health Organization recommendations.Pearson correlation analysis revealed the inter-dependency among elements,and the contribution of each element based on factor loadings showed its percentage contribution compared to others.Water samples from six significant locations were chosen for remediation with three algae:Chlorella vulgaris,Scenedesmus dimorphus,and Phormedium sp.The uptake of pollutants led to the continuous growth of algae during the incubation period of 15 d,effectively removing heavy metals from the river water.The increasing levels of algal counts and the chlorophyll a content confirmed the algal growth during the incubation period,followed by a declining stage after the incubation period.The scanning electron microscopic images of algae before and after the remediation showed no remarkable modification of morphological patterns.This study showed that the uptake of heavy metals using algae is an effective water pollution remediation measure,making the process practicable in the field on a large scale in the near future.

Aqueous extracts of microalgae exhibit antioxidant and anticancer activities

Objective:To investigate the antioxidant and anticancer activities of aqueous extracts of nine microalgal species.Methods:Variable percentages of major secondary metabolites(total phenolic content,terpenoids and alkaloids) as well as phycobiliprotein pigments(phycocyanin, allophycocyanin and phycoerythrin) in the aqueous algal extracts were recorded.Antioxidant activity of the algal extracts was performed using 2,2 diphenyl-1-picrylhydrazyl(DPPH) test and 2,2'azino-bis(ethylbenzthiazoline-6-sulfonic acid(ABTS.) radical cation assay.Anticancer efficiency of the algal water extracts was investigated against Ehrlich Ascites Carcinoma cell(EACC) and Human hepatocellular cancer cell line(HepG2).Results:Antioxidant activity of the algal extracts was performed using DPPH test and ABTS.^+ radical cation assays which revealed 30.1-72.4%and 32.0-75.9%respectively.Anticancer efficiency of the algal water extracts was investigated against Ehdich Ascites Carcinoma Cell(EACC) and Human Hepatocellular cancer cell line(HepG2) with an activity ranged 87.25%and 89.4%respectively.Culturing the promising cyanobacteria species;Nostoc muscorum and Oscillatoria sp.under nitrogen stress conditions(increasing and decreasing nitrate content of the normal BG11 medium,1.5 g/L),increased nitrate concentration(3,6 and 9 g/L) led to a remarkable increase in phycobilin pigments followed by an increase in both antioxidant and anticancer activities in both cyanobacterial species.While the decreased nitrate concentration(0.75,0.37 and 0.0 g/L) induced an obvious decrease in phycobilin pigments with complete absence of allophycocyanin in case of Oscillatoria sp.Conclusions:Nitrogen starvation(0.00 g/L nitrate) induced an increase and comparable antioxidant and anticancer activities to those cultured in the highest nitrate content.

Research on KOH/La-Ba-Al_2O_3 catalysts for biodiesel production via transesterification from microalgae oil

Alumina supports modified by lanthanum （La） and barium （Ba） were prepared by peptization. Catalysts with different KOH contents supported on modified alumina were prepared by impregnation method. Various techniques, including N2 adsorption-desorption （Brunauer-Emmet-Teller method, BET）, X-ray diffraction （XRD）, scanning electron microscopy （SEM）, and fourier transform infrared absorption spectroscopy （FT-IR）. Catalytic activity for microalgae oil conversion to methyl ester via transesterification was evaluated and analyzed by GC-MS and GC. BET results showed that the support possessed high specific surface area, suitable pore volume and pore size distribution. Activity results indicated that the catalyst with 25 wt% KOH showed the best activity for microalgae oil conversion. XRD and SEM results revealed that Al-O-K compound was the active phase for microalgae oil conversion. The agglomeration and changing of pore structure should be the main reasons for the catalyst deactivation when KOH content was higher than 30 wt%.

Fatty acid methyl ester profiles and nutritive values of 20 marine microalgae in Korea

Objecive:To screen the fatty acid(FA) composition of 20 marine microalgae species,including seven Diophyceae,six Bacillariophyeae four Chlorophyceae,two Haptophyceae and one Raphidophyceae species.Methods:Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed.Results:The FA composition of microalgae was speciesspecific.For example,seven different species of Dinophyceae were composed primarily of C14:0,C16:0.C18:0.C20:4n-6.C20:5n-3 and C22:6n-3.while C14:0.C16:0,C16:1.C18:0.C20:5n-3 and C22:6n-3 were abundant FAs in six species of Bacillariophyceae.In addition,four Chlurophyceae,two Haptopkyeeae and one Raphidophyceae species all contained a high degree of C16:1 n-7[(9.2R-34.91)%and(34.48-35.04)%].C14:0[(13.34-25.96)%]and[(26.69-Z8.24)%],and C16:0[(5.89-29.15)%]and[(5.70-16.81)%].Several factors contribute to the nutritional value of microalgae.including the polyunsaturated FA content and n-3 to n-6 FA ratio,which could be used to assess the nutritional quality of microalgae.Conclusions:This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea,and identifies the potential utility of FAs as species-specific biomarkers.

Food processing wastewater purification by microalgae cultivation associated with high value-added compounds production——A review

Microalgae have been considered as an efficient microorganism for wastewater treatment with simultaneously bioenergy and high value-added compounds production.However,the high energy cost associated with complicated biorefinery(e.g.microalgae cultivation,harvesting,drying,extraction,conversion,and purification)is a critical challenge that inhibits its large-scale application.Among different nutrition(e.g.carbon,nitrogen and phosphorous)sources,food processing wastewater is a relative safe and suitable one for microalgae cultivation due to its high organic content and low toxicity.In this review,the characteristic of different food wastewater is summarized and compared.The potential routes of value-added products(i.e.biofuel,pigment,polysaccharide,and amino acid)production along with wastewater purification are introduced.The existing challenges(e.g.biorefinery cost,efficiency and mechanism)of microalgal-based wastewater treatment are also discussed.The prospective of microalgae-based food processing wastewater treatment strategies(such as microalgae-bacteria consortium,poly-generation of bioenergy and value-added products)is forecasted.It can be observed that food wastewater treatment by microalgae could be a promising strategy to commercially realize waste source reduce,conversion and reutilization.

Microalgae:A potential alternative to health supplementation for humans

Microalgae has been consumed in human diet for thousands of years.It is an under-exploited crop for production of dietary foods.Microalgae cultivation does not compete with land and resources required for traditional crops and has a superior yield compared to terrestrial crops.Its high protein content has exhibited a huge potential to meet the dietary requirements of growing population.Apart from being a source of protein,presence of various bio-active components in microalgae provide an added health benefit.This review describes various microalgal sources of proteins and other bio-active components.One of the heavily studied group of bio-active components are pigments due to their anticarcenogenic,antioxidative and antihypertensive properties.Compared to various plant and floral species,microalgae contain higher amounts of pigments.Microalgal derived proteins have complete Essential Amino Acids(EAA)profiles and their protein content is higher than conventional sources such as meat,poultry and dairy products.However,microalgal based functional foods have not flooded the market.The lack of awareness coupled with scarce incentives for producers result in under-exploitation of microalgal potential.Application of microalgal derived components as dietary and nutraceutical supplements is discussed comprehensively.

Microalgae Tolerance to High Concentrations of Carbon Dioxide: A Review

The increasing concentration of carbon dioxide (CO2) in the atmosphere is considered to be one of the main causes of the global warming problem. Moreover, there is an international movement to reduce the emission of CO2 by imposing different measures such as carbon tax. Biological CO2 fixation has been extensively investigated as part of efforts to solve the global warming problem. Microalgae are fast growing systems that can consume high quantities of CO2 to produce different types of biomass. The efficiency of microalgae is highly related to the concentration of CO2 in the growth atmosphere and the higher the concentration of CO2 the better is the growth and hence productivity. The present review aimed at shedding some light upon microalgal capability to sustain their viability and propagate under high CO2 concentration.

Effect of dissolved phosphorus on alkaline phosphatase activity in marine microalgae

Alkaline phosphatase activity (APA) and dissolved phosphorus were monitored during the batch cultures of two bone microalgae. Results indicate that variation of APA was in the shape of 'S' curve. Different specs of dissolved phaphorus had different effects on APA. The concentrations of dis solved inorganic phosphorus (DIP) and and molecular dissolved organic phosphorus (SDOP) had a sig nificant effect on APA, while the concentration of large molecular dissolved organic phosphorus (LDOP) had a little effect on APA., and the increase of APA could accelerate the decomposing of LDOP in the medium. Results also show that algae species and abundance had why a little effect on APA.

Design of a Novel Photobioreactor for Culture of Microalgae

This paper presents the design of a novel photobioreactor for cultivation of microalgae. The body of the reactor with volume of about 40 L is parallelipipedic and divided in five compartments that can be put in series. The optical guides, plunged perpendicularly into the compartments, are upright Plexiglas plates on which side faces there are able to diffuse light laterally and ensure an even distribution of light in the medium. External airlifts through the side columns are used for mixing of culture medium. The external light source is a SON-T lamp mounted inside a projector that provides a conical light dispersion, it is interchangeable and may take different positions. The design offers the photobioreactor characteristics including mainly interchangeable light source, homogenous distribution of light, perfect mixing of suspension of algae, high ratio of illuminating surface to volume of reactor, compactness and absence of contamination. Schemes, view of the photobioractor and data of continuous culture forSpirulina maxima are presented.

Distribution characteristics of planktonic nano－and microalgae in the Prydz Bay and its adjacent Southern Indian Ocean，during austral summer

Nano- and microalgae samples were collected from 34 stations in the Prydz Bay and its adjacent Southern indian Ocean, Antarctica during a Period from December 1990 to January 1991. 122 taxa belonging to 40 genera 5 phyla were identified. Among them diatom occupy 73%,dinoflagellates 20. 5%, the other species 6. 5 %. The average cell abundance of nano- and microalgae was 2551×104 cells/m3 in surface waters. The concentration of nano- and microalgae occurred in the Prydz Bay and its adjacent continental station Ⅳ 7 and northern water area between West Ice Shelf and Shackleton Ice Shelf (Stations Ⅶ 3, Ⅷ 1, Ⅷ 2, Ⅷ 3, Ⅸ 1, Ⅺ 1); and minimal abundance occurred in northwestern area of the Prydz Bay (some stations of section Ⅲ and Station Ⅳ 1-5). The average cell abundance of planktonic nano- and microalgae from net sample was 811. 62×104 cells/m3, and the concentration area occurred in the adjacent continent waters of the Prydz Bay (Stations Ⅳ 2, Ⅳ 4, Ⅳ 5, Ⅳ 7, Ⅴ 4 and Ⅴ 5) and northern area of the West Ice Shelf(Stations Ⅶ 1 and Ⅶ 3); low abundance occurred in eastern area of the Shackleton Ice Shelf (at each station of sections Ⅹ and Ⅺ ). For the vertical distribution of planktonic nano- and microalgae, the maximum value was usually found at the surface and above 50 m depth, and gradually decreased with the water depth from 100 m to 200 m. The relationship between planktonic nano-and microalgal abundance and Euphausia superba density and nutrient (nitrate,phosphate and silicate) contents were negative significance.

Microalgae Cultivation Using Offshore Membrane Enclosures for Growing Algae (OMEGA)

OMEGA is a system for cultivating microalgae using wastewater contained in floating photobioreactors (PBRs) deployed in marine environments and thereby eliminating competition with agriculture for water, fertilizer, and land. The offshore placement in protected bays near coastal cities co-locates OMEGA with wastewater outfalls and sources of CO2-rich flue gas on shore. To evaluate the feasibility of OMEGA, microalgae were grown on secondary-treated wastewater supplemented with simulated flue gas (8.5% CO2 V/V) in a 110-liter prototype system tested using a seawater tank. The flow-through system consisted of tubular PBRs made of transparent linear low-density polyethylene, a gas exchange and harvesting column (GEHC), two pumps, and an instrumentation and control (I&C) system. The PBRs contained regularly spaced swirl vanes to create helical flow and mixing for the circulating culture. About 5% of the culture volume was continuously diverted through the GEHC to manage dissolved oxygen concentrations, provide supplemental CO2, harvest microalgae from a settling chamber, and add fresh wastewater to replenish nutrients. The I&C system controlled CO2 injection and recorded dissolved oxygen levels, totalized CO2 flow, temperature, circulation rates, photosynthetic active radiation (PAR), and the photosynthetic efficiency as determined by fast repetition rate fluorometry. In two experimental trials, totaling 23 days in April and May 2012, microalgae productivity averaged 14.1 ± 1.3 grams of dry biomass per square meter of PBR surface area per day (n = 16), supplemental CO2 was converted to biomass with >50% efficiency, and >90% of the ammonia-nitrogen was recovered from secondary effluent. If OMEGA can be optimized for energy efficiency and scaled up economically, it has the potential to contribute significantly to biofuels production and wastewater treatment.

Experimental studies on dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) production by four marine microalgae

The production of dimethylsulfide （DMS） and dimethylsulfoniopropionate （DMSP） by marine microalgae was investigated to elucidate more on the role of marine phytoplankton in ocean-atmosphere interactions in the global biogeochemical sulfur cycle.Axenic laboratory cultures of four marine microalgae–Isochrysis galbana 8701,Pavlova viridis,Platymonas sp.and Chlorella were tested for DMSP production and conversion into DMS.Among these four microalgae,Isochrysis galbana 8701 and Pavlova viridis are two species of Haptophyta,while Chlorella and Platymonas sp.belong to Chlorophyta.The results demonstrate that the four algae can produce various amounts of DMS（P）,and their DMS（P） production was species specific.With similar cell size,more DMS was released by Haptophyta than that by Chlorophyta.DMS and dissolved DMSP （DMSPd） concentrations in algal cultures varied significantly during their life cycles.The highest release of DMS appeared in the senescent period for all the four algae.Variations in DMSP concentrations were in strong compliance with variations in algal cell densities during the growing period.A highly significant correlation was observed between the DMS and DMSPd concentrations in algal cultures,and there was a time lag for the variation trend of the DMS concentrations as compared with that of the DMSPd.The consistency of variation patterns of DMS and DMSPd implies that the DMSPd produced by phytoplankton cells has a marked effect on the production of DMS.In the present study,the authors’ results specify the significant contribution of the marine phytoplankton to DMS（P） production and the importance of biological control of DMS concentrations in oceanic water.

Cultivation of microalgae for biodiesel production:A review on upstream and downstream processing

Fluctuating market price of fossil fuel and overwhelming emission of greenhouse gases to the atmosphere have resulted in climate change and have been a global concern in this decade. Hence, biodiesel has become an alternative option to fossil diesel as it is renewable and environmentally friendly. Nevertheless, this alternative fuel that is usually derived from terrestrial oil crops will cause shortage in food supply and deforestation if mass production is realized. In recent years, cultivation of aquatic microorganism(particularly microalgae) to produce biodiesel is considered as a practical solution due to their high growth rate and ability to synthesize large quantity of lipid within their cell. However, the development of energy and cost-efficiency of microalgae cultivation system are the main issues in producing renewable microalgae biodiesel. Of late, wastewater or organic compost has been used as the cultivation medium as it can provide sufficient nutrients to sustain microalgae growth.Microalgae cultivation method and system are vitally important as these factors undoubtedly affect the final microalgae biomass and lipid yield. In this review, the cultivation system of microalgae, nutrients demanded for microalgae production, cell harvesting and drying, microalgae oil extraction, and utilization of microalgae biomass for biodiesel production are introduced and discussed. It is anticipated to convey clearer perspectives in upstream and downstream processes in microalgae-derived biodiesel production.

Biodiesel Production from <i>Spirulina-Platensis</i>Microalgae by <i>In-Situ</i>Transesterification Process

This research investigates the effect of reaction variables that strongly affect the cost of biodiesel production from non-edible Spirulina-Platensis microalgae lipids, and use the acid-catalyzed in situ transesterification process. Experiments were designed to determine how variations in volume of reacting methanol, the concentration of an acid catalyst, time, temperature and stirring affected the biodiesel yield. The total lipid content of Spirulina-Platensis microalgae was obtained to be 0.1095g/g biomass. The weight of the by-product glycerol obtained was used to predict the percentage yield conversion of microalgae oil biodiesel. Best results (84.7%), a yield of fatty acid methyl ester (FAME), were obtained at 100% (wt./wt.oil) catalyst concentration, 80 ml methanol volumes, 8 h reaction time and 65℃ reaction temperature with continuous stirring at 650 rpm. Properties of the produced biodiesel were measured according to EN 14214 standards.

Heavy Metal Bioaccumulation and Toxicity with Special Reference to Microalgae

The bioaccumulation and toxicity of heavy metals were reviewed with special reference to microalgae, the key compo- nent of the food web in aquatic ecosystems. Heavy metals enter algal cells either by means of active transport or by endocytosis through chelating proteins and affect various physiological and biochemical processes of the algae. The toxicity primarily results from their binding to the sulphydryl groups in proteins or disrupting protein structure or displacing essential elements. Metals can break the oxidative balance of the algae, inducing antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX). The amount of oxidized proteins and lipids in the algal cells thus indicates the severity of the stress. Algal tolerance to heavy metal is highly dependent upon the defense response against the probable oxidative damages. Pro- duction of binding factors and proteins, exclusion of metals from cells by ion-selective transporters and excretion or compartmen- talization have been suggested with regard to reducing heavy metal toxicity. However, a comprehensive description on the mecha- nisms underlining metal toxicity of microalgae and gaining tolerance is yet to be elaborated.

Biomineralization of Zinc-Phosphate-Based Nano Needles by Living Microalgae

Up to now, chemical synthesis routes only provide restricted opportunities for the formation of structured nano particles. In contrast, living microorganisms generate nano materials of well defined shapes by the precise control of biomineralization. Here we reveal new principles for the generation of functional nano materials through the process of biomineralization. We used the detoxification mechanism of the unicellular alga Scenedesmus obliquus to generate a techno logically interesting zinc-phosphate-based nano material. The algae were incubated in media with a sublethal zinc concentration (6.53 mg Zn dm-3) for 4 weeks. Using BF-and ADF-STEM imaging combined with analytical XEDS we could show that nano needles containing phosphorus and zinc were formed inside the living cells. Further more, the cells incubated with zinc show a strong fluorescence. Our findings indicate that the algae used polyphosphate bodies for detoxification of the zinc ions, leading to the generation of intracellular zinc-phosphate-based nano needles. Beside the technological application of this material, the fluorescent cells can be used for labeling of e.g. biological probes. This new experimental protocol for the production of an inorganic functional material can be applied also for other substances.

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

Calcium carbonate (CaCO3) occurs in the three polymorphs calcite, aragonite and vaterite. The formation of these crystals in inorganic solutions is influenced by parameters like pH, temperature or impurities. Living freshwater microalgae can also induce the formation of CaCO3 when they live in a suitable environment containing saturated amounts of Ca2+. Through this biologically induced biomineralization only the formation of the polymorph calcite has been reported yet. We investigated the precipitates which have been formed in solutions containing the freshwater microalgae Scenedesmus obliquus and different zinc amounts (0, 3.27 and 6.53 mg Zn2+/l) by XRD and SEM. As references precipitates from the same solutions but without algae were investigated. We could show that the presence of living microalgae has a great influence on the precipitation of calcium carbonate crystals. In algae-containing media without or with a low zinc amount always calcite and aragonite are formed. In the corresponding medium with 6.53 mg Zn2+/l pure aragonite crystals were built. In contrast, in the inorganic, algae-free solutions without zinc, pure calcite is precipitated. Both inorganic solutions with zinc show major calcite precipitation and weak aragonite precipitation. Thus the algae cells advance significantly the formation of aragonite, which is enhanced by the presence of zinc cations in the media. Possible mechanisms are discussed.