Prospects of a hot spring-originated novel cyanobacterium, Scytonema ambikapurensis, for wastewater treatment and exopolysaccharide-enriched biomass production.

The present work performs the polyphasic characterization of a novel cyanobacterial species Scytonema ambikapurensis isolated from an Indian hot spring and evaluates its wastewater bioremediation potential. While the physicochemical analyses of the wastewater indicated high load of nutrients and metals, the wastewater bioremediation experiment performed using the test cyanobacterium denoted the removal of 70 and 86% phosphate, 49 and 66% sulfate, 96 and 98% nitrate, 91 and 92% nitrite, 95 and 96% ammonia, 66 and 72% chloride, 79 and 81% zinc, 68 and 80% nickel, 81 and 90% calcium, and 80 and 90% potassium from the autoclaved and un-autoclaved wastewater, respectively, after 20 days of culturing. The kinetics study of zinc and nickel removal from wastewater revealed that the cyanobacterium employed sequential biosorption (by following pseudo-second-order kinetics model) and bioaccumulation methods to remove these two metals. The quality of the autoclaved and un-autoclaved wastewater was further improved by the cyanobacterium through reduction of hardness by 74 and 81%, respectively. In wastewater, the cyanobacterium not only enhanced its biomass, chlorophyll and carbohydrate contents, but also produced small amount of released and high capsular exopolysaccharide (EPS). The FTIR and TGA analyses of capsular EPS unraveled that it was a negatively charged sulfated biomolecule having thermostability up to 240 °C, which suggested its possible use as excellent emulsifying, viscosifying, and biosorption agent. The credibility of this EPS as biosorption agent was ascertained by evaluating its metal chelating ability. Finally, the experimental data denoting the ability of S. ambikapurensis to bioremediate wastewater and simultaneously produce EPS was statistically validated by PCA1-pollutant removal model and the PCA2-cellular constituent model, respectively. Briefly, the study discloses that the cyanobacterium has huge biotechnological and industrial importance as it bioremediates wastewater and simultaneously produces thermostable exopolysaccharide.

Decoding whole genome of Anoxybacillus rupiensis TPH1 isolated from tatapani hot spring, India and giving insight into bioremediation ability of TPH1 via heavy metals and azo dyes.

A moderately thermophilic, gram-positive genomospecies Anoxybacillus rupiensis TPH1 was isolated from Tatapani hot spring, Chhattisgarh, India. Genome of 3.70 Mb with 42.3% GC subsumed 4131 CDSs, 65 tRNA, 5 rRNA, 35 AMR and 19 drug target genes. Further, comparative genomics of 19 Anoxybacillus spp. exhibited an open pan genome of 13102 genes along with core (10.62%), unique (43.5%) and accessory (45.9%) genes. Moreover, phylogenomic tree displayed clustering of Anoxybacillus spp. into two distinct clades where clade A species harbored larger genomes, more unique genes, CDS and hypothetical proteins than clade B species. Further, distribution of azoreductases showed FMN-binding NADPH azoreductase (AzoRed1) presence in clade A species only and FMN-binding NADH azoreductase (AzoRed2) harboring by species of both clades. Heavy metal resistance genes distribution showed omnipresence of znuA, copZ and arsC in both clades, dispersed presence of cbiM, czcD, merA and feoB over both clades and harboring of nikA and acr3 by few species of clade A only. Additionally, molecular docking of AzoRed1, AzoRed2, ZnuA, CopZ, Acr3, CbiM, CzcD, MerA and NikA with their respective ligands indicated high affinity and stable binding. Conclusively, present study provided insight into gene repertoire of genus Anoxybacillus and a basis for the potential application of this thermophile in bioremediation of azo dyes and heavy metals.

Deciphering the mechanisms of zinc tolerance in the cyanobacterium Anabaena sphaerica and its zinc bioremediation potential.

Cyanobacteria adopt a variety of changes at proteomic and metabolic levels for surviving under harmful environmental conditions including heavy metal stress. The current study investigates the impact of zinc stress on the proteome of Anabaena sphaerica to get an insight into its molecular mechanisms of zinc tolerance. The study revealed three different aspects that were associated with the zinc tolerance in A. sphaerica: (i) the reduced expression of photosynthesis, nitrogen fixation, energy metabolism, respiratory, and transcriptional/translational proteins probably to conserve energy and utilizing it to sustain growth; (ii) the enhanced expression of metallothionein and ferritin domain protein All 3940 to chelate free zinc ions whereas upregulation of antioxidative proteins for detoxifying reactive oxygen species; and (iii) the expression of large numbers of hypothetical proteins to maintain the important cellular functions. Furthermore, over expressions of sulfate adenylyl transferase and cystathionine beta synthase along with the increased synthesis of peptidases and thiolated antioxidant proteins were also noticed which denoted cysteine synthesis under sulfur deprivation possibly by mobilizing the sulfur from dead cells and its channelization towards the production of thiolated antioxidants. Besides tolerating excess amount of zinc, A. sphaerica exhibited high zinc biosorption efficiency which confirmed its outstanding zinc bioremediation potential.

Biotic elicitor-induced changes in growth, antioxidative defense, and metabolites in an improved prickleless Solanum viarum.

Solanum viarum serves as a raw material for the steroidal drug industry due to its alkaloid and glycoalkaloid content. Elicitation is well-known for measuring the increase in the yield of bioactive compounds in in vitro cultures. The current study was performed for the accumulation of metabolites viz. solasodine, solanidine, and α-solanine in S. viarum culture using microbial-based elicitors added in 1%, 3%, 5%, and 7% on 25th and 35th day of culture period and harvested on 45th and 50th days of culture cycle. The treatment of 3% Trichoderma reesei and Bacillus tequilensis culture filtrate (CF) significantly increased biomass, alkaloids/glycoalkaloid content, and yield in S. viarum. T. reesei was found to be the best treatment for enhanced growth (GI = 11.65) and glycoalkaloid yield (2.54 mg DW plant-1) after the 50th day of the culture cycle when added on the 25th day. The abundance of gene transcripts involved in the biosynthesis of alkaloids/glycoalkaloids, revealed by quantitative real-time PCR expression analysis correlates with the accumulation of their respective metabolites in elicited plants. Biochemical analysis shows that elicited plants inhibited oxidative damage caused by reactive oxygen species by activating enzymes (superoxide dismutase and ascorbate peroxidase) as well as non-enzymatic antioxidant mechanisms (alkaloids, total phenols, total flavonoids, carotenoids, and proline). The findings of this study clearly demonstrate that the application of T. reesei and B. tequilensis CF at a specific dose and time significantly improve biomass as well as upregulates the metabolite biosynthetic pathway in an important medicinal plant- S. viarum. KEY POINTS: • Biotic elicitors stimulated the alkaloids/glycoalkaloid content in S. viarum plant cultures. • T. reesei was found to be most efficient for enhancing the growth and alkaloids content. • Elicited plants activate ROS based-defense mechanism to overcome oxidative damage.

Degrading ability and robust antioxidative defence system led to SDS tolerance in cyanobacterium Fischerella sp. lmga1.

To test the tolerance and degradation potential of the cyanobacterium Fischerella sp. lmga1 for surfactant, sodium dodecyl sulfate (SDS), different doses of SDS (10, 30, 40, 50, 70 and 100 µM) were used for the growth. The lower doses of SDS supported the growth of cyanobacterium whereas the higher doses were found to be inhibitory but the cyanobacterium somehow managed its survival up to 100 µM SDS. However, a significant reduction was observed in the pigment and protein content. A substantial accumulation of carbohydrate at 70 µM SDS may act as an osmoprotectant for the survival of the cyanobacterium. The higher doses of SDS also triggered the ROS generation and lipid peroxidation which showed negative impact on the PSII efficiency. Simultaneously, an efficient ROS mitigation system (SOD and CAT activity) has also been worked up to 70 µM SDS while APX was enhanced only up to 50 µM SDS. Furthermore, the SDS degrading potential was investigated and almost 80% of the SDS was degraded after 6th days of treatment in the cyanobacterium. Hence, the results suggested that due to robust antioxidative defence system and ability to degrade the surfactant this cyanobacterium showed significant tolerance toward SDS.

Physiological mechanisms of aluminum (Al) toxicity tolerance in nitrogen-fixing aquatic macrophyte Azolla microphylla Kaulf: phytoremediation, metabolic rearrangements, and antioxidative enzyme responses.

To investigate the extent of aluminum toxicity tolerance of eco-friendly, fast-growing, fresh water, pteridophytic Azolla-Anabaena symbiotic association in terms of altered physiological signals; Azolla microphylla Kaulf was exposed to 0 (control), 100, 250, 500, and 750 µM AlCl3, at pH 4.5 for 6 days. The adversity of Al was increased in a dose-dependent manner and the highest was recorded at 750 µM AlCl3. Despite the significant loss in membrane integrity (80% electrolyte leakage) due to an enhanced generation of H2O2, A. microphylla reflected only 50% growth inhibition (fresh and dry weight) at 500 µM AlCl3 (LD50). However, the average root length of Azolla was drastically reduced at high concentration due to their direct contact with aluminum-containing growth medium. Contrary to this, the whole association maintained moderate chlorophyll, carbohydrate content, photosynthetic efficiency, nitrogen-fixing ability, and nitrogen content at high Al concentration. Probably, growth protection was pertained through significant detoxification of H2O2 by employing an efficient antioxidative defense system including antioxidative enzymes (SOD, APX, and CAT) and non-enzymatic antioxidant carotenoids. An enhanced level of phenolics and flavonoids in the root exudates possibly maintained a non-toxic level of aluminum inside the cell (195.8 µg Al/g FW) which makes A. microphylla a suitable pteridophytic plant to not only remove toxic Al from the contaminated sites but also to improve nitrogen status of those regions. Graphical abstract ᅟ.

Phylogeny and evolutionary genetics of Frankia strains based on 16S rRNA and nifD-K gene sequences.

16S rRNA and nifD-nifK sequences were used to study the molecular phylogeny and evolutionary genetics of Frankia strains isolated from Hippöphae salicifolia D. Don growing at different altitudes (ecologically classified as riverside and hillside isolates) of the Eastern Himalayan region of North Sikkim, India. Genetic information for the small subunit rRNA (16S rRNA) revealed that the riverside Frankia isolates markedly differed from the hillside isolates suggesting that the riverside isolates are genetically compact. Further, for enhanced resolutions, the partial sequence of nifD (3' end), nifK (5' end) and nifD-K IGS region have been investigated. The sequences obtained, failed to separate riverside isolates and hillside isolates, thus suggesting a possible role of genetic transfer events either from hillside to riverside or vice versa. The evolutionary genetic analyses using evogenomic extrapolations of gene sequence data obtained from 16S rRNA and nifD-K provided differing equations with the pace of evolution being more appropriately, intermediate. Values of recombination frequency (R), nucleotide diversity per site (Pi), and DNA divergence estimates supported the existence of an intermixed zone where spatial isolations occurred in sync with the temporal estimates. J. Basic Microbiol. 2015, 54, 1-9.

Decoding cyanobacterial phylogeny and molecular evolution using an evonumeric approach.

Forty-one heterocystous cyanobacteria strains representing 12 cyanobacterial genera collected from all across India were assessed in phylogenetic and evolutionary perspectives. The structural gene 16S ribosomal RNA (rRNA) and the functional genes nifD and psbA were selected as molecular chronometers in this study. The phylogenetic analyses demonstrated the monophyly of heterocystous cyanobacteria with significant intermixing, along with establishing the polyphyly of Stigonematales, strongly supporting the need for re-amendments in cyanobacterial taxonomy and systematics. Molecular trends obtained did not clearly reflect the phenotypic affiliations, thus advocating for genetic characterizations using more molecular markers. Large-scale evonumeric extrapolations of gene sequence data of all the three molecular markers was performed to assess the evolutionary pace of heterocystous cyanobacteria on the basis of nucleotide diversity, recombination frequencies, and the DNA divergence between the sampled taxa. The obtained results tilted the evolutionary pace in favor of the less complex Nostocales thus indicating that possibly the simple non-branched forms are more flexible and adaptive for evolutionary diversifications as compared to the more complex and branched ones. This study hence represents a unique blend of molecular phylogeny with evogenomic sequence analyses for understanding the genetic diversity, phylogeny, and evolutionary pace within the heterocystous cyanobacteria.

Molecular phylogeny, population genetics, and evolution of heterocystous cyanobacteria using nifH gene sequences.

In order to assess phylogeny, population genetics, and approximation of future course of cyanobacterial evolution based on nifH gene sequences, 41 heterocystous cyanobacterial strains collected from all over India have been used in the present study. NifH gene sequence analysis data confirm that the heterocystous cyanobacteria are monophyletic while the stigonematales show polyphyletic origin with grave intermixing. Further, analysis of nifH gene sequence data using intricate mathematical extrapolations revealed that the nucleotide diversity and recombination frequency is much greater in Nostocales than the Stigonematales. Similarly, DNA divergence studies showed significant values of divergence with greater gene conversion tracts in the unbranched (Nostocales) than the branched (Stigonematales) strains. Our data strongly support the origin of true branching cyanobacterial strains from the unbranched strains.

Sodium transport and mechanism(s) of sodium tolerance in Frankia strains.

The mechanism(s) underlying differential salt sensitivity/tolerance were investigated in the terms of altered morphological and physiological responses against salinity such as growth, electrolyte leakage, Na⁺ uptake, efflux, accumulation and intracellular concentrations of macronutrients among the Frankia strains newly isolated from Hippöphae salicifolia D. Don. Growth was minimally reduced at 500 and 250 mM NaCl respectively in HsIi10 and rest of the strains (HsIi2, HsIi8, HsIi9) which proved that 500 and 250 mM NaCl are the critical concentrations for the respective strains. The differences in the sodium influx/efflux rate was responsible for the differential amount of remaining sodium among the frankial strains and might be one of the primary determinants for the reestablishment of macronutrients (Mg²âº, Ca²âº and K⁺) during salinity. Secondly, the interactive effect of sodium influx/efflux rate, remaining sodium and intracellular macronutrients (Mg²âº, Ca²âº and K⁺) concentration has been responsible for the extent of membrane damage and growth sustenance of the tolerant/sensitive frankial strains during salinity. HsIi10 showed better co-regulation of various factors and managed to tolerate salt stress up to considerable extent. Therefore, HsIi10 can serve as a potential biofertilizer in the saline soil.

Phylogenetic comparison among the heterocystous cyanobacteria based on a polyphasic approach.

Phylogenetic comparison has been done among the selected heterocystous cyanobacteria belonging to the sections IV and V. The hierarchical cluster analysis based on antibiotics sensitivity showed a distant relationship between the members of Nostocales and Stigonematales. Thus, multiple antibiotic resistance pattern used as marker provide easy, fast, and reliable method for strain discrimination and genetic variability. However, morphological, physiological (both based on principal component analysis) and biochemical analysis grouped true branching cyanobacteria along with the members of section IV. Molecular analysis based on 16S rRNA gene sequences revealed that Hapalosiphon welwitschii and Westiellopsis sp. were grouped in cluster I whereas Scytonema bohnerii, a false branching genera showed a close proximity with Calothrix brevissima in cluster II. Cluster III of clade 2 included Nostoc calcicola and Anabaena oryzae which proved the heterogeneity at the generic level. Cluster IV the largest group of clade 2 based on 16S rRNA gene sequences includes six strains of the genera Nostoc, Anabaena, and Cylindrospermum showing ambiguous evolutionary relationship. In cluster IV, Anabaena sp. and Anabaena doliolum were phylogenetically linked by sharing 99% sequence similarity. Probably, they were of the same genetic makeup but appear differently under the diverse physiological conditions. Section IV showed polyphyletic origin whereas section V showed monophyletic origin. Results suggested that either morphological or physiological or biochemical or molecular attribute is not sufficient to provide true diversity and phylogeny of the cyanobacteria at the generic level and thus, a polyphasic approach would be more appropriate and reliable.

Chemotaxonomy of heterocystous cyanobacteria using FAME profiling as species markers.

The fatty acid methyl ester (FAME) analysis of the 12 heterocystous cyanobacterial strains showed different fatty acid profiling based on the presence/absence and the percentage of 13 different types of fatty acids. The major fatty acids viz. palmitic acid (16:0), hexadecadienoic acid (16:2), stearic acid (18:0), oleic acid (18:1), linoleic (18:2), and linolenic acid (18:3) were present among all the strains except Cylindrospermum musicola where oleic acid (18:1) was absent. All the strains showed high levels of polyunsaturated fatty acid (PUFAs; 41-68.35%) followed by saturated fatty acid (SAFAs; 1.82-40.66%) and monounsaturated fatty acid (0.85-24.98%). Highest percentage of PUFAs and essential fatty acid (linolenic acid; 18:3) was reported in Scytonema bohnerii which can be used as fatty acid supplement in medical and biotechnological purpose. The cluster analysis based on FAME profiling suggests the presence of two distinct clusters with Euclidean distance ranging from 0 to 25. S. bohnerii of cluster I was distantly related to the other strains of cluster II. The genotypes of cluster II were further divided into two subclusters, i.e., IIa with C. musicola showing great divergence with the other genotypes of IIb which was further subdivided into two groups. Subsubcluster IIb(1) was represented by a genotype, Anabaena sp. whereas subsubcluster IIb(2) was distinguished by two groups, i.e., one group having significant similarity among their three genotypes showed distant relation with the other group having closely related six genotypes. To test the validity of the fatty acid profiles as a marker, cluster analysis has also been generated on the basis of morphological attributes. Our results suggest that FAME profiling might be used as species markers in the study of polyphasic approach based taxonomy and phylogenetic relationship.

Diversity of Frankia strains nodulating Hippophae salicifolia D. Don using FAME profiling as chemotaxonomic markers.

Twelve Frankia strains isolated from Hippophae salicifolia D. Don or Alnus glutinosa or Comptonia peregrine, showed the significant variation in fatty acid composition viz. palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidic acid (20:0) and erucic acid (22:1) suggesting the strain specific variability among the Frankia strains. Presence of Erucic acid (22:1), a major component of the oil obtained from the seeds of Brassica sp., albeit in lesser amount in the few studied frankial strains, is the first report. Cluster analysis on the basis of fatty acid composition suggests the presence of two distinct clusters with similitude coefficient ranging from 0.75 to 1.00. Cluster I with HsIi2 showed great divergence from other 11 frankial strains (Cluster II). The two sub groups were distinguished in cluster II: IIa contained five strains isolated from H. salicifolia and these strains are distantly related to the strains of cluster IIb isolated from different host. There is high degree of similarity among the frankial strains of Cluster IIb which suggests that the frankial strains might be evolved from the same ancestor. FAME profiling might be useful tool in the study of polyphasic approach based taxonomy and phylogenetic relationship.

Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don.

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains.