SUSTAINABLE BIOFUEL PRODUCTION FROM ESTUARINE DIATOMS

Purpose of the study: Microalgae with better carbon sequestration potential, higher biomass, and lipid productivity in comparison to terrestrial counterparts is emerging as a viable sustainable alternative to fossil fuels. Diatoms, a subgroup of a broader category of microalgae are well-known for their role as a bio-indicator in palaeo-climatological studies across historical timelines. Understanding ecology, community structure, and habitat preference of diatoms are prerequisites for prioritizing diatom strains towards sustainable biofuel production along with value-added product extraction. Method: Selection of appropriate strains, economically viable harvesting and environmentally sound transesterification are the challenges faced in microalgal industry. The present study focusses on understanding the variability in benthic diatom community assemblages across seasons and its affinity to different substrata at fixed sampling locations in an estuarine eco-system through field sampling for twelve months covering all seasons. Main Findings: The results highlight the tolerance and resilience in select diatom strains in fluctuating water qualities and seasonal variations, the insights much needed during mass cultivation under open environments. Statistical data analyses revealed distinct demarcation between sensitive and tolerant species with selective habitat preferences and resilience to fluctuating environmental conditions. Variations of benthic diatom community structure across various substrata highlights substratum affinity of diatom strains. From the inferences derived through field studies, mixed consortia of diatoms were developed, monitored, and optimized for higher biomass and lipid productivity under controlled laboratory conditions. Application of the Study: The results highlight the prospects of phyco-prospecting of indigenously grown diatom strains for sustainable biofuel production.


INTRODUCTION
Benthic diatoms are highly sensitive to hydrogeological and environmental changes in the water body. Diatom community structure generally varies concerning seasons and land-based discharges with associated nutrient fluctuations to the system in the form of run-offs and point-source pollutions. Understanding diatoms and its community structure have been useful in aquatic health assessment and cost-effective eco-system monitoring studies during the last few decades. Diatoms have aided as bioindicators of environmental changes like eutrophication, metal contamination, salinization, acidification, and catchment alterations with land-use changes (Dixit et al., 1992). Seasonal variability at the regional scale and level of anthropogenic inputs influences community structure in lotic eco-systems, especially the estuaries (Saranya et al., 2018). Anthropogenic inputs include land-based discharges that alter nutrient levels of the aquatic eco-systems in its immediate vicinity. Benthic diatoms are sensitive to such impulsive habitat changes in terms of physico-chemical and hydrological parameters. Hence understanding the variations in community structure at different locations with varied anthropogenic stress levels across seasons would give insights on species preference to a particular substratum and its resilience to fluctuating environmental conditions. Screening based on this criterion would help in the selection of season tolerant strains for biofuel production. In recent decades, there has been a greater convergence towards sustainable bioresource utilization especially for biofuel production given addressing the country's energy shortages. Dwindling stock of fossil fuels, fluctuating prices in the world oil market, and concurrence among sensible nations to reduce CO2 levels necessitates focus towards renewable energy. Global biofuel production has risen by 2.6% in 2016, which is higher by 0.4% than 2015's average (BP World Renewable Energy Statistics report 2016). As per the IEA statistical report (2016), the world has relied up to 4% on bioenergy derived liquid transportation fuels and this Figure is expected to increase by 4.3% in 2020. There are numerous global endeavors, evident from the annual publication of 550 research papers after 2005 (Azadi et al., 2017). These endeavors highlight the interest in attaining energy self-reliance and energy security.
Fossil fuel-dependent electric power generation and petroleum products driven transportation sectors especially in India significantly contribute to the greenhouse gas footprint (Ramachandra and Shwetmala, 2012; Ramachandra and Shwetmala, 2009; Ramachandra and Hegde, 2015). Bioethanol and biodiesel are two primary liquid biofuels that are gaining momentum in the country's energy scenario. Lignocellulosic biomasses and seaweeds are considered for bioethanol production, while microalgae are considered as significant lipid resources for biodiesel production. The algae constitute third-generation biofuel feedstock. Figure 1 represents the share of different algal species (family-wise) experimented so far for biofuel production. A major share in the exploitation of algae as lipid precursors is from green microalgae than diatoms to produce biofuel. Diatoms belonging to the class Bacillariophyceae have a prominent advantage in terms of its ubiquitous presence and are benthic with substratum attachment characteristics, which would considerably lower the cost associated with microalgae harvesting.
Diatoms have proven biofuel characteristics, evident from prioritizing 40 diatom strains (out of 50 microalgae strains) with higher lipid potential among the total 3000 screened strains by Aquatic Species Program hosted by the US Department of Energy (Sheehan et al., 1998). However, the economic viability of microalgae-based biofuel production systems is a major challenge in commercializing diatoms-based biofuels. Hence it is a rationale to choose a diatom strain or a consortium of strains that are consistently productive during all seasons under fluctuating environmental conditions, then a strain that gives higher productivity only under optimal conditions (Hildebrand et al., 2012). Prioritizing potential strains entails understanding respective strains/species' ecological preference under varying field conditions.

LITERATURE REVIEWS
Estuaries are transition zones or biologically productive ecotones with a constant confluence of fresh and saline waters (Kennish, 2002). They act as filters/buffer zones that trap natural and anthropogenic pollution causing materials between continents and pelagic environments (Qiu et al., 2010). They are dynamic water bodies characterized by strong gradients of salinity and nutrients due to land-based discharges (Lohrenz et al., 1990). Estuaries experience seasonal variations in irradiance, temperature, nutrients, and freshwater inflow, which influences the microalgal community structure (Seguro et al., 2015). Notable contributions to benthic diatom community structure as a part of aquatic health assessment programs in eco-system monitoring investigations are now available (Bere, 2016;Dalu and Froneman, 2016;Hering et al., 2006;Kelly et al., 1998;Reid et al., 1995;Reid, 2005;Schoeman, 1979;Tan et al., 2017;Townsend and Gell, 2005). Diatoms are highly resilient group of micro-organisms Ramachandra et al., 2011) with higher productivity, that dominates the world oceans with approximately 40% of the marine primary productivity and 20% of global carbon fixation (Hildebrand et al., 2012;Leblanc et al., 2018;Levitan et al., 2014). Nearly one-fifth of world's photosynthesis is carried out by diatoms (Armbrust, 2009;Nelson et al., 1995). Diatoms comprise of the lowest level marine food web and are responsible for export of carbon from surface to deeper oceans (Tréguer et al., 2018). Moreover, Diatoms gain importance at myriad dimensions as nano-fabricators of biogenic silica, progenitor elements of present day's crude petroleum reserves (Levitan et al., 2014), biofuel precursors, live feed for aquaculture, bio-active compound synthesizers, bio-based silicon semiconductors for solar photovoltaics (Jeffryes et al., 2011;Zhu et al., 2013) eminent source of carotenoids and essential fatty acids, subject of clue in the field of criminology and forensic sciences (Sun et al., 2015). Diatoms not only are the sources of lipids but also has potential scope for value added products due to the presence of high value bio active compounds such as carotenoids, pigments, neuro-excitatory amino acid derivative toxindomoic acid and Naviculana sulphated polysaccharide having antiviral property (Prestegard et al., 2009).

RESEARCH PROBLEM
The study focusses on understanding the habitat wise ecological characteristics and diatom community structure. This not only aids in the selection of promising diatom species but also helps in understanding respective environmental conditions (which helps in the design of sustainable bioreactors). Environmental gradients and seasonal fluxes in water and biological parameters were monitored monthly for a period of one year to understand the influence of physico-chemical parameter and nutrient dynamics on benthic diatom community structure.
This study also intended to capture the seasonal variability with respect to substrata and habitat specificity of benthic diatoms and its relation to community structure. Insights into ecological perspectives would also lower the costs involved in the optimization of growth parameters. Seasons and environmental gradients play a pivotal role in richness, abundance, and community structure of diatoms especially in benthic diatoms -the most predominant group of all micro-organisms in the intertidal zones of the estuary. As benthic diatoms adhere to substrata and form biofilms with the help of exo-cellular polysaccharide secretion, understanding substratum specificity could give additional insights on the type of community structure and the habitat preferences with substratum specificity of sensitive and tolerant species.

AIM OF THE STUDY
The objectives of the present study are to: i.
Understand the effect of seasonal fluctuations of water quality on diatom species composition; ii.
Investigate the variations in benthic diatom community structure in terms of tolerance and sensitivity through statistical analyses; and iii.
Estimate the biomass and lipid potential of enriched diatom from a particular substratum that had shown the highest species composition during ecological monitoring studies.

Study Area
Benthic diatoms were sampled across salinity gradients along the shorelines (intertidal regions) of the Aghanashini estuary extending between 14° 27' 53.  The water along with diatom samples was collected at select sampling locations in the intertidal regions of the estuary, once in every month, during July 2016 to June 2017 at low tide hours of the day as visibility and better accessibility of landmass, mud-flats, sediments, and stones was possible during low tide as the water recedes towards the downstream during low tide. Water and benthic diatom samples were collected from different substrata. Physico-chemical parameters like air temperature, water temperature, pH, DO and salinity were measured at respective sampling locations. Dissolved oxygen was estimated through Wrinkler's titration method following APHA 2005 protocol. Nutrient levels (Nitrates (NO3 -), Phosphates (PO4 3-) and silicates (SiO4 4-) of the water samples was assessed in the laboratory within 4 hours of sampling, following the standard protocols of APHA (2005).

Diatom Sample Collection and Preparation
Diatom samples were collected at each sampling locations (from substrata such as stones, cobbles, boulders, macrophytes, sedges, shells, halophytes (mangrove plantations), sediments, and sand. Triplicate samples were collected from each substratum. The epilithic (diatoms attached to stones) were collected by scraping using a clean spatula after washing the substrata under the water to remove the silt deposited on hard surfaces. Epiphytic (diatoms attached to plants) were collected mainly from small mangrove plants grown along the shoreline. A portion of sedges and the mangrove stems were cut and placed inside the sampling bottle containing distilled water and were shaken to dislodge the diatom cells attached on the surface of the plants. Epipelic (Diatoms attached to sediments) and Episammic (diatoms attached to sand) were collected from damp and moist surfaces exposed during low tide by moving the sampling container along the sand/sediment surface. The collected diatom samples were immediately subjected to fixation using 2% Lugol's Iodine to cease cell multiplication and preserved at 4 o C until analysis. Table 1 lists the details of study locations and different substrata considered at each station during the study. Diatom samples collected were pre-treated and processed to remove the organic contents inside the cells using potassium permanganate and hydrochloric acid as per the standard protocols of ( Patrick and Reimer, 1966). The identified species were also enumerated as per DARES protocol by counting 400 valves per sample to determine the relative abundances, species diversity, and community composition. Light micrographic images of few diatom species were listed in Figure 3.

Statistical Analyses
Two-way ANOVA statistical analysis was carried out to estimate the level of significance of seasonal physico-chemical parameters on species richness (S). The data were compiled season-wise (pre-monsoon, post-monsoon and monsoon) and subjected to agglomerative hierarchical clustering for understanding the dynamics of diatom species' abundance w.r.t seasons. Non-metric Multi-Dimensional Scaling (MDS) technique was used to understand the species abundance pattern of benthic diatom communities' season-wise across stations. Agglomerative hierarchical clustering and nMDS analysis were performed using R Studio version 1.1.423. Season wise and station wise diatom diversity (Shannon -Wiener (H')) and Simpson's Dominance (D) were computed using Shannon diversity and Simpson's dominance indices. Overall species richness (SR) and species abundance (SA) with respect to different substrata sampled at each station are illustrated in the form of a pie chart. The species that had shown a relative abundance of >15% and recorded consistently during all three seasons were considered as tolerant species and radar plot was generated using season-wise relative abundances of those tolerant species at each study station.  The water quality results depicted the seasonal variability. Air temperature showed wider fluctuations primarily due to higher insolation during pre-monsoon seasons followed by reduced levels due to precipitation. Air and water temperature variations were comparable to earlier studies in tropical estuaries of the south-west coast of India (Madhu et al., 2007;Martin et al., 2008). pH values in water samples in all sampling locations across seasons were neutral to slightly basic (7.88 ± 0.04) with no significant fluctuations. pH of a waterbody is mainly influenced by induced anthropogenic disturbances in the form of domestic or industrial effluents. Variations in pH were minimal as the study locations were mainly oligotrophic to mesotrophic. The salinity during monsoon season in central and upper reaches (stations S1 -S4) was lower (5.6 ± 2.58 ppt) compared to lower reaches (stations S5 and S6) with (18.1 ± 2.9 ppt). A marked wider salinity gradient was observed in all the stations during different seasons. The upper (S1 and S2) and the middle reaches (S3 and S4) with an influx of freshwater during monsoon had freshwater regime. However, the salinity levels peaked to 30 ppt with the onset of the summer season (pre-monsoon). The salinity levels were distinctly different across stations during post-monsoon with low salinity levels (6.25 ± 2.47 ppt) in upper reaches, mid salinity levels (18.12 ± 2.29 ppt) in central reaches and high salinity (25.37 ± 1.59 ppt) in lower reaches of the estuary.

Variations in Hydrological Parameters
Nutrient levels across the stations were more of temporal dilutions due to anthropogenic inputs. There was no trend in nutrient variation across seasons among the stations and the results were highly location specific and prone to non-point source pollutions. Higher nitrate levels were observed (5.12 ± 0.57 µg/L) during pre-monsoon season followed by post-monsoon

Substrata wise Species Richness and Abundance
The data recorded substrata wise for a period of one year were consolidated to understand the role of substrata in species assemblage ( Figure 7). The results demonstrated the highest species abundance (36%) at S2 followed by S3 (31%). Station S6 showed the least species abundance (4%). Stations S4 and Station S5 contributed to 11% and 5% of species abundance respectively. Among the different substrata considered, sediments had maximum epipelic diatom species richness of 40, 36, and 28 respectively at stations S2, S3, and S4. Higher species richness in sediments was followed by stones/rocks (Epilithons) at almost all the stations. Epiphytons attached to plant substrata had the third-highest species richness in S2, S3, and S4 stations with species richness of 35, 21, and 23 respectively, which is similar to the earlier report (Townsend and Gell, 2005). Lower species richness and poor community structure are due to the grazing pressure. Episammons (diatoms growing on sand) had the least species richness in polyhaline lower reaches stations S5 and S6 with S = 12 and S = 18, due to persistent wave action, washing away the community leading to lower diatom affinity in that substrata.

Species Tolerance
The presence of a species, its abundance as well as its distribution is largely determined by their level of tolerance to abiotic environmental factors. Tolerance is the ability or endurance of a species to survive in hostile environmental conditions. When a species is exposed to prolonged unfavorable conditions, the species tends to build up resilience to withstand harsh environmental conditions and in turn evolve as a tolerant species having higher ability to survive under wider ranges of fluctuating conditions, which is observed in certain diatom species with the consistent presence in fluctuating environmental conditions (seasonal variability). These tolerant species abundances w.r.t different seasons at each station is illustrated in the form of a radar plot in Figure 8. The species present during all the three seasons with their relative abundances greater than 15% were considered as "tolerant species" capable of surviving fluctuating environmental conditions and their season-wise relative abundances were plotted, which illustrates the site-specific abundances of tolerant species in premonsoon, postmonsoon and monsoon. The graph was generated by clubbing the tolerant species to the genus level.  Overall tolerant species' abundance was highest during pre-monsoon, followed by post-monsoon and the least abundance in monsoon season. During premonsoon, the species abundance of Cyclotella meneghiniana at station S2 was the highest (74.5%) followed by consortia of Navicula sp. (Navicula lanceolata, Navicula bicapitata, Navicula forcipata, Navicula mutica and Navicula amphisbaena) with 68.9% at station S3. Polyhaline (S5) station showed a dominance of Melosira spp. with corresponding species abundance of 72.7% during the premonsoon season. Though the abundance of these species showed a marked peak during pre-monsoon season of the year, the presence of those species was recorded at other seasons as well with varying abundance levels, proving the species to be tolerant with evolved ecological adaptations to season-based dynamism prevailing in real-time estuarine systems.
Season wise analyses of species' relative abundances reveal higher relative abundance during pre-monsoon with >15% of tolerant species followed by post-monsoon and the least relative abundances were observed during monsoon season (due to influx of freshwater). As per the tolerance results primarily determined by their presence in almost all the seasons with their corresponding species-wise relative abundances to be >15%, the diatoms belonging to the genus of Achnanthes spp., Nitzschia spp., Navicula spp., Cyclotella spp., Melosira spp. were found to be tolerant with consistent availability at different stations as well as during different seasons.
Among the pennate ones, the genus belonging to Amphora and Nitzschia are known to have pronounced heterotrophic capabilities (Linkins, 1973;Werner, 1977). Most of the Nitzschia sp. is known to be obligate heterotrophs with their habitat preferences in strong heterotrophic environments like decaying piles of seaweeds, with little or nil light penetration. Cyclotella meneghiniana was also known to have strong heterotrophic capabilities (Horner and Alexander, 1972;Lewin and Hellebust, 1976;Lylis and Trainor, 1973). Another dominant taxon observed very frequently in lower reaches (S5 and S6) was known to be a taxon frequently encountered in higher insolation regions, especially in tidal pools and upper intertidal regions. This taxon was proven to be relatively tolerant of inter-tidal exposure to desiccation (Castenholz, 1963;Stevenson et al., 1996) which could have given Achnanthes sp. the ability to survive at higher insolation over large boulders subjected to prolonged intertidal exposure. Melosira sp. could dominate in varying pH conditions and warmer waters with higher light intensity. It is found that Navicula sp. which occurs mostly over the sediment surfaces and on other aquatic plants are facultative heterotrophs that utilize organic matter found in the substrate (Werner, 1977)., which justifies dominance of certain species across different seasons in more than one station.

Clustering based on Species Abundance
Month-wise species composition along with its relative abundance of diatoms was organized season-wise and subjected to agglomerative hierarchical clustering ( Figure 9) to understand the species predominance and season sensitivity. Cluster analysis resulted in three distinct clusters based on their species abundance across different seasons. Cluster 1 formed the major group of diatom species that are sensitive and predominantly either freshwater species or euryhaline species.
The presence of which is observed only in specific sites at a particular season with its relative abundance <2%. Cluster 1 includes freshwater species such as Cocconeis placentula, Aulacoseira granulata and salinity tolerant euryhaline species such as Amphiprora alata, Eunotia flexuosa, Spermatogonia sp., Pleurosigma marcum etc., with least abundance recorded in one of the three seasons. Cluster 2 formed the second major group with a moderate species abundance of >5%. Cluster 3 formed the dominant groups with a higher relative species abundance of 8% -15%. Out of 86 different species of diatoms, more than 80% of the population were either recorded only in upstream (less saline) regions during monsoon periods or only at downstream regions during pre/post-monsoon seasons forming cluster 1. Those species could be collectively termed as sensitive species, the presence or absence of which is influenced by one or more of the environmental parameters and even the slightest change in those parameters could have led to the species turnover by tolerant species. The species recorded in the upstream regions were found to exhibit predominant freshwater habitat characteristics. The species that exhibited such characteristics include Synedra ulna, Aulacoseira granulata, Bacillaria paradoxa, Cocconeis sp. Gomphonema sp., Cymbella sp., Pinnularia sp., and Diploneis sp. (Appendix 1). The presence of freshwater species was confined to upstream and middle estuarine portions in monsoon season. On the other hand, species like Biddulphia laevis, Licomophora sp., Amphiprora alata, Epithema argus were found only in downstream (S5 and S6) stations of the estuary. Hence these species could be considered as salinity loving/euryhaline species and their absence during monsoon and post monsoon could be attributed to overall low salinity of the estuary during those two seasons.

Season-wise Variation in Species Composition
Non-metric multidimensional scaling analysis was performed to understand the dynamics of the diatom community structure among stations by considering season-wise species abundance across each station ( Figure 10). NMDS is a rank-based approach in which the ordination is achieved through an iterative process based on pairwise dissimilarity in a low dimensional space. Here NMDS is plotted considering species abundance summed up season-wise to understand the commonness in species across stations. The plot shows ordination hulls formed with respect to pre-post-and monsoon seasons across stations (S1 PRM -S6 PRM), (S1 M -S6 M), and (S1 POM -S6 POM). The ordination hulls generated season-wise, which demarcates tolerant species that are present throughout the year from station-specific sensitive species.

Tolerant Species for Bioremediation and Biofuel
Recent research on third-generation biofuel production is increasingly concentrating on using wastewater as a source of nutrients for the growth of microalgae. If a biofuel industry is to be set up based on open/raceway ponds using wastewater as a source of nutrients, running the industry at consistent productivities throughout the year proves challenging as contamination control becomes quite difficult in such open systems. The introduction of monoculture strains or sensitive species further complicates the scenario as the pond is continuously exposed to both diurnal and seasonal variations in weather as well as water quality used for biomass enrichment. Hence, phyco-prospecting consortia of tolerant species with year-round availability irrespective of microclimate/environmental conditions would be a better choice in averting the risks of contamination or crash encountered in monoculture sensitive strain-based cultivation systems. Thus, from the results of the present study, it is apt to consider tolerant diatom or consortia of tolerant diatoms capable of exhibiting robust growth despite fluctuations and variations in the environmental factors that greatly influence its growth. The species having potential for phyco-prospecting evident from the results of the present study are Amphora sp., Nitzschia sp., Navicula sp., Achnanthes sp., Melosira sp., Coscinodiscus sp., and Cyclotella sp. The above-mentioned species when chosen as a candidate strain or as consortia of strains for phyco-prospecting has better chances of survival in open pond/raceway pond systems exhibiting enhanced biomass productivities.

Lipid Potential of Select Tolerant Diatom Strains
Microalgae are widely considered as biodiesel feedstocks due to its better lipid productivity and shorter cycling period with quick multiplication rates when compared to conventional terrestrial oil seeds.  (Fields and Kociolek, 2015). These results corroborate the prospects of select tolerant species as potential biofuel strains.

Laboratory Experiments to Elucidate the Lipid Potential of Indigenous Diatom strains
Benthic diatoms were collected from rocks from one of the study sites (S4) and immediately inoculated in F/2 media under sterile conditions. The nutrient enrichment provided by F/2 media facilitated a mixed consortium of diatoms to grow in the culture with a working volume of 250 mL. The light intensity was maintained at 90 µmol m -2 s -1 and the temperature was maintained at 29 ± 2 o C. The salinity of the cultures was maintained at 28 psu. The nutrient-enriched mixed cultures were then analyzed for species composition variations throughout the growth period as well as its lipid content once the cell reached its stationary phase. The consortium consisted of Coscinodiscus sp., Nitzschia panduriformis, Melosira sp., Navicula sp., Skeletonema sp., Amphora sp., Camphylodiscus sp., and Nitzschia obtusa. Figure 11 represents the changes in the relative abundance of each species in the consortium during the complete growth period of ten days. After ten days, the diatoms were harvested and subjected to lipid extraction using a modified Folch method.
The culture consortium was further sub-cultured as aliquots under various treatments such as nitrogen and silicon starvation to understand the percentage increase of lipids as dry cell weights with respect to no treatment (control) sample. Results on varying treatments revealed that nitrogen starvation had exhibited the highest lipid content (17 -25% based in terms of dry cell weight). The biomass yield was found to be 80 -160 mg/L. The nitrogen starved algal biomass extracted lipids were further subjected to acid catalysed transesterification to obtain FAMEs. The FAME thus obtained were analysed using GC-MS to understand the types of fatty acids produced by the diatom consortia. Figure 12 represents the peaks showing variations (%) in diatom derived FAMEs. Palmitic acid methyl ester was the dominant fatty acid recorded followed by palmitoleic acid, myristic acid, and trans-vaccenic acid methyl ester. These results elucidate the potential of indigenous diatom strains consortium in the production of third-generation biofuels.

CONCLUSIONS
Understanding ecological characteristics and habitat preferences of microalgae are crucial to avoid contamination issues in large scale microalgal production systems, especially while using wastewater as a source of nutrient for algal growth. , Season tolerant strains would ensure year-round microalgal biomass productivity and reduce the effort as well as additional operating costs involved in eliminating species turn over. This research has provided valuable insights pertaining to the seasonal dynamics of diatoms along an estuarine shoreline of Indian west coast in-order to screen tolerant diatoms species over sensitive ones. Amphora sp., Nitzschia sp., Navicula spp., Achnanthes sp., Melosira sp., Coscinodiscus sp., and Cyclotella sp. have better tolerance to seasonal variations in water quality with potential to phyco-prospecting these indigenous strains.

RECOMMENDATION
Screening a diatom or consortia of dominant diatom species capable of surviving in rugged environmental conditions, could become ideal candidates for third-generation biofuel cultivation system on a regional basis, especially suitable for tropical countries like India. Lab-scale results on indigenously isolated mixed consortia of diatoms reinstate its potential and prospects of diatoms becoming valuable feedstock for third generation biofuel production as well as value-added products.