Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus.

Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce enzymes that metabolize this polymer. In this work we have demonstrated the presence of cellulolytic activity in the supernatant of Scenedesmus quadricauda cultures and we identified the presence of extracellular cellulases in the genome of five Scenedesmus species. Scenedesmus is a green alga which grows in both freshwater and saltwater regions as well as in soils, showing highly flexible metabolic properties. Sequence comparison of the different identified cellulases with hydrolytic enzymes from other organisms using multisequence alignments and phylogenetic trees showed that these proteins belong to the families of glycosyl hydrolases 1, 5, 9, and 10. In addition, most of the Scenedesmus cellulases showed greater sequence similarity with those from invertebrates, fungi, bacteria, and other microalgae than with the plant homologs. Furthermore, the data obtained from the three dimensional structure showed that both, their global structure and the main amino acid residues involved in catalysis and substrate binding are well conserved. Based on our results, we propose that different species of Scenedesmus could act as biocatalysts for the hydrolysis of cellulosic biomass produced from sunlight.

Current Regulatory Requirements for Biosimilars in Six Member Countries of BRICS-TM: Challenges and Opportunities.

Background: The aim of the study was to identify, interpret, and compare the current perspectives of regulatory agencies in six member countries of BRICS-TM (Brazil, Russia, India, China, South Africa, Turkey, and Mexico) on the different criteria used for biosimilar development and marketing authorisation process. Methods: A semi-quantitative questionnaire was developed covering the organisation of agency, biosimilar development criteria and marketing authorisation process and sent to seven regulatory agencies covering the BRICS-TM countries. All data was kept anonymous and confidential. Data processing and analysis was carried out; descriptive statistics were used for quantitative data and content analysis was employed to generate themes for qualitative data. Results: Out of the seven regulatory agencies included in the study, six representatives provided the responses. The perspectives of these six regulatory agencies varied on a number of aspects relating to the review criteria for biosimilar development and licencing process. The most prevalent model for data assessment is the "full review" of a marketing authorisation application. There is lack of a standard approach across the agencies on sourcing of the reference biological product, in vivo toxicity studies and confirmatory clinical studies. Most agencies restrict interaction with biosimilar developers and any scientific advice is non-binding. The marketing authorisation approval depends on scientific assessment of the dossier, sample analysis and GMP certification. The agencies do not issue any public assessment report specifying the summary basis of biosimilar approval. Conclusion: Regulatory agencies across the six emerging economies are steadily improving the regulatory mechanism in the area of biosimilars. However, there remains scope for increasing the effectiveness and efficiency of the processes by encouraging open and transparent interaction with developers, adopting a flexible approach toward accepting advanced analytical data in lieu of clinical studies and enhancing regulatory reliance amongst agencies. This will help to simplify the new biosimilar development programmes and make them more cost-effective.

Bioprospecting microalgae from natural algal bloom for sustainable biomass and biodiesel production.

Natural algal bloom consists of promising algal species which could be a feasible option for the source of bulk biomass and biodiesel production. It has been found in five natural fresh water algal blooms (Uttar Pradesh, India), containing high nitrogen (N) (4.6 ± 0.32 mg/L) and phosphorus (P) (4.12 ± 0.29 mg/L) concentration during spring (23.9-25.9 °C) and summer season (32.0-35.0 °C). Among the isolated algae from naturally occurring bloom, Chlorella sorokiniana MKP01 exhibited highest biomass (1.02 ± 0.02 g/L) and lipid content (174.1 ± 9.6 mg/L) in untreated tap water and urea/single super phosphate (SSP) in the ratio (2:1). The biodiesel quality was assessed and found to be with the Indian and international standards. Algal bloom was artificially developed in the open pond containing 10,000 l tap water supplemented with Urea/SSP (2:1) for a consistent supply of bulk biomass, yielded 8 kg of total biomass and lipid 1.3 kg.

Draft genome sequence and detailed characterization of biofuel production by oleaginous microalga Scenedesmus quadricauda LWG002611.

BACKGROUND: Due to scarcity of fossil fuel, the importance of alternative energy sources is ever increasing. The oleaginous microalgae have demonstrated their potential as an alternative source of energy, but have not achieved commercialization owing to some biological and technical inefficiency. Modern methods of recombinant strain development for improved efficacy are suffering due to inadequate knowledge of genome and limited molecular tools available for their manipulation. RESULTS: In the present study, microalga Scenedesmus quadricauda LWG002611 was selected as the preferred organism for lipid production as it contained high biomass (0.37 g L-1 day-1) and lipid (102 mg L-1 day-1), compared to other oleaginous algae examined in the present study as well as earlier reports. It possessed suitable biodiesel properties as per the range defined by the European biodiesel standard EN14214 and petro-diesel standard EN590:2013. To investigate the potential of S. quadricauda LWG002611 in details, the genome of the organism was assembled and annotated. This was the first genome sequencing and assembly of S. quadricauda, which predicted a genome size of 65.35 Mb with 13,514 genes identified by de novo and 16,739 genes identified by reference guided annotation. Comparative genomics revealed that it belongs to class Chlorophyceae and order Sphaeropleales. Further, small subunit ribosomal RNA gene (18S rRNA) sequencing was carried out to confirm its molecular identification. S. quadricauda LWG002611 exhibited higher number of genes related to major activities compared to other potential algae reported earlier with a total of 283 genes identified in lipid metabolism. Metabolic pathways were reconstructed and multiple gene homologs responsible for carbon fixation and triacylglycerol (TAG) biosynthesis pathway were identified to further improve this potential algal strain for biofuel production by metabolic engineering approaches. CONCLUSION: Here we present the first draft genome sequence, genetic characterization and comparative evaluation of S. quadricauda LWG002611 which exhibit high biomass as well as high lipid productivity. The knowledge of genome sequence, reconstructed metabolic pathways and identification of rate-limiting steps in TAG biosynthesis pathway will strengthen the development of molecular tools towards further improving this potentially one of the major algal strains for biofuel production.

Cell growth kinetics of Chlorella sorokiniana and nutritional values of its biomass.

The present study investigates the effects of different physico-chemical parameters for the growth of Chlorella sorokiniana and subsequently determination of nutritional values of its biomass. Most suitable temperature, light intensity, pH, and acetic acid concentration were 30°C, 100 µmol m(-2)s(-1), pH 7.5, and 34.8mM, respectively for the growth of this microorganism. Arrhenius growth activation energy, Ea was calculated as 7.08 kJ mol(-1). Monod kinetics constants: maximum specific growth rate (µ max) and substrate (acetic acid) affinity coefficient (Ks) were determined as 0.1 ± 0.01 h(-1) and 76 ± 8 mg L(-1), respectively. Stoichiometric analysis revealed the capture of 1.83 g CO2 and release of 1.9 g O2 for 1g algal biomass synthesis. Algal biomass of C. sorokiniana was found rich in protein and several important minerals such as Mg, Ca, and Fe. Astaxanthin and ß-carotene were extracted and quantified using high performance liquid chromatography.

Development of suitable photobioreactors for CO2 sequestration addressing global warming using green algae and cyanobacteria.

CO(2) sequestration by cyanobacteria and green algae are receiving increased attention in alleviating the impact of increasing CO(2) in the atmosphere. They, in addition to CO(2) capture, can produce renewable energy carriers such as carbon free energy hydrogen, bioethanol, biodiesel and other valuable biomolecules. Biological fixation of CO(2) are greatly affected by the characteristics of the microbial strains, their tolerance to temperature and the CO(2) present in the flue gas including SO(X), NO(X). However, there are additional factors like the availability of light, pH, O(2) removal, suitable design of the photobioreactor, culture density and the proper agitation of the reactor that will affect significantly the CO(2) sequestration process. Present paper deals with the photobioreactors of different geometry available for biomass production. It also focuses on the hybrid types of reactors (integrating two reactors) which can be used for overcoming the bottlenecks of a single photobioreactor.

Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo.

Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polkappa, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.