Novel nitrogen-fixing bacteria isolated from the root nodules of the Pea plant

Rhizobia is a large group of bacteria developing symbiotic relation with their host plants by forming root nodules and fixing atmospheric nitrogen. The bacteria belonging to this group are important from agriculture and environmental perspective. Three strains of rhizobia were collected from Indian Trans-Himalaya region, isolated from root nodules of Pea plant. The characterization was done using 16S rRNA, atpD and recA genes. Based on 16S rRNA gene sequencing, isolated strains showed ≥99.9% sequence similarity to the members within the Rhizobium leguminosarum group. Phylogenetic analysis based on sequences of atpD, recA and 92 core genes indicated that two of the three strains were closely related to the group Rlaguerreae while one strain was closely related to the Rhidalgonense group. Two strains closely related to the group R. laguerreae have 60.8 mol% of DNA G+C content. Based on characterization studies, these two strains represent a novel species, Rhizobium indicum sp.nov. The study provided a detailed taxonomic species description of these novel species.

The nodC and nifH genes play an important role in nodulation and nitrogen fixation respectively. These genes were amplified for all three strains which confirmed their genetic potential for symbiosis.  All three strains have nodC gene sequence similar to the strains of Rhizobium leguminasarum that belong to the symbiovar viciae and isolated from different countries.

Genome studies revealed that the complete genome, chromosome and plasmids of these strains are comparable in size to the other strains of Rhizobium leguminasarum. Researchers also examined the genome sequences of the two strains to identify the compliment of nod genes in these strains. Both strains contained a cluster of 12 nod genes. Phylogenetic analysis revealed that both these strains are closely related to Rhizobium laguerreae followed by Rruizarguesonis and R. leguminosarum.

Morphological analysis revealed that the strains are rod-shaped, gram-negative and motile. Colonies were translucent, cream-white and convex. A total of 20 different cellular fatty acids in different quantities were detected in these three strains. Overall, the fatty acid compositions of the newly isolated strains were identical to the type strains of Rhizobium leguminosarumRhizobium laguerreae and Rhizobium ecuadorense, with differences in their proportions. The strain were positive for oxidase and catalase.

To conclude, both the novel strains are closely related to each other and can be differentiated from other species of the genus Rhizobium by their housekeeping gene sequences, ANI values, genome nucleotide analysis, and phenotypic and chemotaxonomic features.


National Centre for Microbial Resource- An emerging Microbial Resource Centre of India

Microbes are tiny organisms which are not visible to the naked eyes. Microbes contribute to different phenomenon like global climate change, energy generation, production of bio-molecules for pharmaceuticals and other industries, plant nutrition and soil health. Microbes are also good source of enzymes, vitamins, amino acids and help in production of food, fuel and fodder. Microbes play an important role in biotechnology research and are essential for development of many products and processes. Recent studies revealed that huge microbial diversity of immense biotechnological potential is yet to be cultured. In order to increase the contribution of microbial resources towards world’s bio-economy the cultivation, taxonomical characterization and bio-banking of microbes is essential. Establishment of a quality microbial resource centre (MRC) with state-of-the-art facilities is essential for safeguarding microbial diversity and for developing bio-economy of the nation.

National Centre for Microbial Resource (NCMR) affiliated with National Centre for Cell Science, Pune is a project under Department of Biotechnology, Government of India. NCMR is well equipped with all the essential facilities fulfilling the criteria of a good Microbial Resource Centre (MRC). The centre has well trained researchers and technicians with extensive experience in cultivation, preservation, biosafety, metagenomic, genomics, proteomics, intact microbiome preservation and handling capacity of different group of organisms including anaerobes, photoautotrophs, fungi, archaea, cyanobacteria etc. NCMR is actively involved in research related to different areas of microbiology including ecology and systematics.

Cultivation, characterization and preservation of microbes and supplying them for academic and industrial usage is the main aim of the centre. Cultivation and accession of microorganisms of environmental, agricultural, medicinal and biotechnological importance to researchers and industries will be contributing to the sustainable development of India. NCMR also provides different services including deposit services, culture supply services, identification/characterization services, genomics/metagenomic services etc. The centre has newly launched different services like genome-based taxonomy, imaging by scanning electron microscopy, phytoplasma detection, testing for microbial load for food safety certifications and deposit of AMR strains. NCMR continuously tries to establish new facilities and services.

Some microorganism are pathogenic and possess environmental and personal risk. Along with providing Good Lab Practices (GLP) and Biosafety Levels (BSL) trainings, centre also tries to lower the risk of transmission, contamination and spread of pathogens in the environment. NCMR is actively involved in supplying microorganisms to national and international research communities, industries and academic institutions. NCMR is establishing a unique repository of microorganisms with extended antimicrobial resistance (AMR) traits and simultaneously working on generation of Indian standard strains for research related to development of antibiotic resistance in pathogens. The centre is also aiming at establishment of animal and human cell line repository for patent processed under Budapest Treaty, WIPO.

Observation of data based on publications, offered services, and deposition etc. indicates that NCMR is performing well as one of the Microbial Resource Centre of the nation. Due to extensive agricultural, medicinal and environmental potential microbes are the key component of bio-based economy of any nation. Researchers in different parts of the world are working towards cultivation, characterization and exploitation of microbial diversity to boost their bio-based economy. Considering the available biodiversity and microbial resources establishment of Microbial Resources Centre (MRC) with state-of-the-art facility is current requirement of developing nations.


Methods for Microbiome preservation

A. Cell Alive System (CAS) Technique for Intact Microbiome Preservation
The Cell Alive System (CAS), is a novel technique primarily developed to improve the preservation method in food industry, especially for preservation of raw seafood items. In this technique, the spinning motion of water during freezing prevents formation of crystal lattices. The presence of alternating or oscillating magnetic field and mechanical vibrations in this system induces uniform cooling and minimum size ice crystal formation in preserved sample. This process prevents cellular and molecular damage to the preserved sample. CAS freezing technology has been approved as a well-optimized tool for long-term preservation of deep-sea sediment samples for geo-microbiological studies. With suitable improvements to this methodology, researchers might be able to achieve more storage time and viability.

B. Cryopreservation and Lyophilization in Microbiome Preservation
Cryopreservation and lyophilisation are well-known methods for long-term preservation of microbial cultures. Cryoprotectants are a group of chemicals, which prevent the ice crystal formation during freezing process. Also, ultra-low temperature gives a high stability and viability to the microbial cells. Glycerol and DMSO are commonly used cryoprotectants. However, Galacto-oligosaccharides (GOS) another cryoprotectant is getting more attention due to its prebiotic properties. Different microbes behave differently in terms of viability and stability with different cryoprotectants and more study is needed to be done in this area.

C. Gelatine Disk Method: Preservation of Sample During Transportation
This is a simple bacterial preservation technique using gelatine disk. This method gives good result for successful preservation of a number of pathogens but, did not perform well for prolonged preservation of Vibrio and Neisseria species. Gelatine disk method allows survival of fastidious organisms up to one year at – 20 C storage. There is no direct report on the use of the gelatine disk method for the conservation of an intact microbiome, but due to its ability to preserve the viability and functional stability of a variety of bacterial strains, researchers may consider this technique to be a potential alternative to long-term microbiome preservation.

D. Cellular Immobilization or Entrapment
Cellular immobilization or entrapment in the gelling matrix is another alternative for long-term preservation of microbial viability and functionality. Formation of micro-droplets or beads with different gelling agents like alginate and Acacia-gum is well known method of microbial cell entrapment. This method is getting good response in probiotic industry due to better survival and functionality of the entrapped cells compared to other preservation methods and sustained release in gut after intake. Organisms of probiotic importance like Bifidobacterium and Enterococcus have been preserved using this method. Adding cryoprotectants and antioxidants to the gelling matrix ensures long-term survival and stability of entrapped cells. This method is used with pure culture of microbes however like other preservation methods it can be explored for mixed microbiome preservation, and protocols needs to be optimized.

F. Electrospinning and Electrospraying (Microencapsulation) in Microbiome Preservation
Many microbial cells are sensitive to micro-environmental changes and it’s difficult to preserve them using normal preservation techniques. Microencapsulation method works better for such sensitive microbes. Electrospinning and Electrospraying are novel microencapsulation techniques used to maintain the viability and functionality of microorganisms. For Electrospinning, the viscosity of the polymer is high. Polymer solution in Electrospraying is less visocus, offering nano- or microscale fibres of polymer entrapped microbial cells and small droplets (beads) respectively. Unlike other methods, non-involvement of temperature in these processes reduce the protein- denaturation from harsh temperature conditions and consequently increase the cellular viability and functionality during preservation. Since both these methods are used for small groups of microbes, further studies are required to test its preservation efficiency for other group of microbes.


Treatment of industrial effluents and analysis of their impact on the structure and function of microbial diversity in a unique process

Excessive nutrients released from factories along with pollutants are the key reason for the dense growth of plants in water bodies, which poses a serious environmental problem in many developed and developing countries. The dairy and fertilizer industries produce a large amount of wastewater loaded down with organics and nutrients including phosphate and nitrate. Most of the wastewater treatment plants treat these two nutrients in different procedures in separate setups, which is time consuming and expensive. Nitrogen contamination is being treated by nitrification and denitrification processes whereas phosphate in the wastewater is treated by enhanced biological phosphorus removal (EBPR).

EBPR process is preferred for phosphorus treatment but this method was found to be inefficient for removing nitrogen entities. In one of the previous studies, a low oxygen phase was added to the conventional EBPR method for treating nitrogen entities. But it made the process more complicated and expensive.

In this study, researchers have developed a process known as anoxic-aerobic single-cell Sequential Batch Reactor (AnASBR) which have many benefits over the conventional and the modified EBPRs. This process is capable of treating the industrial effluent polluted with nitrate along with phosphate and organics. Also this process has the potential to change the diversity as well as the metabolic adaptation of the involved microorganism radically than that of the conventional process.

In order to help AnASBR as a long-term and safe alternative for the treatment of wastewater rich in nutrients, this study aimed at achieving the following.
I. Optimization of the AnASBR to achieve maximum removal of nutrients and Chemical Oxygen Demand (COD).
II. Treatment of two industrial effluents in the optimized AnASBR
III. In-depth study of microbial diversity and putative metabolic potential of the microbes.

Two sequential batch reactors (SBR) with Anoxic-Aerobic process (AnAP) were established for the treatment of effluent from two industries; phosphate fertilizer (AnASBR_PPL) and dairy industry (AnASBR_DW). Up to 90% and ~80% of COD removal were achieved in AnASBR_PPL and AnASBR_DW, respectively. Interestingly change in influent had an impact on bacterial diversity. All reactors were filled from the parent SBR with the same deposit, while the population remained largely unchanged, the population’s evenness changed drastically to get accustomed to the changing climate. Rhodocyclales was accounted for 66% of the population in AnASBR-PPL and 22% in AnASBR-DW. Rhodocyclales was widely reported as the significant phosphate accumulator in full-scale EBPR systems.

AnASBRs observed the predominance of Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. The identity of the core group members remained relatively constant at steady-state activity, but their relative abundances in both reactors changed considerably as a result of varying industrial effluent. However, population of few strains such as Lactobacteriales, Enterobacteriales changed drastically with respect to the influent, as these strains were predominant in AnASBR_DW but not present in AnASBR_PPL.

A novel Anoxic-Aerobic Process (AnAP) that eliminated the anaerobic cycle was designed and worked for the simultaneous remediation of industrial effluent phosphate, nitrate, and chemical oxygen demand (COD). COD and nutrients in the effluents were successfully remediated simultaneously in the optimized reactors. Partial sludge granulation was observed in both SBRs which makes it easier for the microbes to consume a very small amount of phosphate and nitrate under aerobic conditions. The metagenomic study revealed denitrifying phosphate accumulating organisms (DNPAOs) as the dominant group of bacteria in both the reactors. PAOs were also present in the consortium but not in dominance. Diverse micro-flora ensured robustness and performance stability in the process of high strength and various industrial effluent treatments. This study proved the efficiency of the new AnASBR as an alternative system that is energy efficient with higher ease of operation for the treatment of real industrial effluents without fail.


Novel bacterial species isolated from the Rann of Kachchh, India

-By Kranti Karande

The genus Rhizobium is a large group of bacteria, and most species are known for their symbiotic fixation of nitrogen within the root nodules of leguminous plants. Rhizobium is a major genera in the Rhizobiaceae family. Presently, this genus comprises 90 recognized species. Non-symbiotic and free-living Rhizobium members have been found in different types of soils.

Rann of Kachchh is reputed to be the world’s largest salt desert. The temperature of this desert goes upto 50 degree celsius during summers and drops below zero degree celsius during winters. Due to the hot and hyper saline environment, there is a vast possibility of identifying novel microbes with high economic and industrial potential in this region. A bacterial strain was isolated during investigations on the bacterial diversity of the saline desert soil obtained from Kachchh Rann, India.

For phylogenetic analysis, the 16 S rRNA based sequencing technique was used. Genomic, physiological, and chemotaxonomic approaches were used to analyze the strain. The API 20E and API ZYM systems and BIOLOG GN III systems were used to understand biochemical characteristics, enzyme activities, and oxidation / reduction of carbon sources.The genotypic and phenotypic data generated for this strain revealed that the strain represents a novel species of the genus Rhizobium, for which Rhizobium desertarenae sp. nov. name is proposed.

The cells of this strain were rod-shaped, gram-negative, and non-motile. At 28 degrees Celsius and pH 7.0 the strain grows well and can tolerate up to 2 percent NaCl. It is oxidase and catalase positive. Based on 16S rRNA gene phylogeny, the strain belongs to the genus Rhizobium, with the highest similarity to Rhizobium wuzhouense and Rhizobium ipmoeae. The average nucleoide identity of this strain was less than 82%, to members of the family Rhizobiaceae. The genomic DNA G+C content is 58.6%. This strain showed differences in physiological, phenotypic and protein profiles estimated by MALDI-TOF MS analysis to its closest relatives.


Comparative genomics of Whooping cough vaccine strains from India

-By Kranti Karande

File:Bordetella pertussis on Charcoal Agar supplemented with ...

Pertussis also known as Whooping cough is a highly contagious respiratory disease, endemic in all countries. It is caused by the bacterium Bordetella pertussis that survives in mouth, nose and throat region. Pertussis is known for uncontrollable, frequent coughing and breathing difficulties, a disease found most dangerous in infants.

The aim of pertussis vaccination is to reduce risk of severe disease in infants and young children. There are two types of pertussis vaccines: whole-cell vaccines based on killed B.pertussis organisms and acellular pertussis dependent on one or more highly purified pertussis antigens. Despite high vaccine coverage, re-emergence of pertussis is observed globally. There are different factors contributing for this disease resurgence. Genetic divergence in the circulating strains of B.pertussis has been reported as one of the important contributing factors for the same.

Our current knowledge of the genetic evolution of B.pertussis in circulating strains is largely focused on studies carried out in countries using ACVs (Acellular Vaccines) targeting only a few antigens used in the production of ACVs. In order to better understand adaptation to vaccine-induced selection pressure, it will be essential to study B.pertussis populations in developing countries where WCVs (Whole-Cell Vaccines) are used. India is a significant user and global supplier of WCVs.

This article briefly describes a study conducted by researchers to compare genomes of B.pertussis vaccine strains and clinical isolates reported from India. Genetic divergence was mostly studied in circulating strains of B. pertussis concerning vaccine antigens such as pertussis toxin, pertactin, fimbriae and filamentous hemaglutinnin. Whole genome sequences obtained from five different vaccine strains were compared with reference strain (Tohama-I) and two recently isolated clinical isolates from India. Core-genome based phylogenetic analysis was also performed using isolates reported from countries using ACV.

Whole-genome analysis of vaccines and clinical isolates reported from India revealed high genetic similarity and conserved genome among strains. Phylognetic analysis showed that clinical and vaccine strains share genetic closeness with reference strain.

This study provides detailed characterization of vaccine and clinical strains reported from India, which will further facilitate epidemiological studies on genetic shifts in countries which are using WCVs in their immunization program.


Why Lonar lake in Maharashtra turned pink?

Lonar lake, also known as Lonar crater, is a national monument of geological heritage, saline, soda lake located in Maharashtra India. This lake is believed to have been formed after a meteorite hit the Earth some 50,000 years ago.

This lake came to the news in the second week of June due to a sudden change of water color to pink. For nature enthusiasts as well as scientists, this color change was curious. Dr. Yogesh Shouche at NCMR-NCCS Pune explained the possible reason behind this color change.

” Clear water appears blue in color, the change in water color can happen due to dissolved minerals or because of microbial activity”, said Dr. Shouche. Lonar Lake is rich in microbial diversity including archaea, fungi, bacteria , and viruses. We had very little knowledge about the microbial composition of Lonar Lake ten years ago but now we have much better information about the microbes in this water body. Scientists are studying this ecosystem and it consists of a variety of microorganisms.

Algae are present in this water body. Red algae, Trichodesmium erythraeum, mainly produces a pigment called phycoerythrin. The increase in salinity of water due to stressful conditions could have led to an excess production of phycoerythrin pigment, which could have led to a change in color.
Dunaliella salini is type of halophile green algae especially found in salt water bodies. Under stressful conditions, this algae produces beta carotene, red-orange pigment. Beta carotene is present in carrots, which is the reason behind carrots color red.

Haloarchaea and halobacteria are found in oceans or lakes with high salt content. The studies done by scientists at NCMR-NCCS Pune have reported presence of different haloarchaea at Lonar lake. Bacteriorhodopsin and bacteriorubrin are the pigments produced by haloarchaea which impart pink color.

So whether the change in color of Lonar lake is unique or something unusual? The answer is NO.

Such phenomenon is observed at different water bodies all over the world. Lake Hilier in Australia is found to be pinkish in color all over the year. Hut laggoons in Australia, Natron lake in Tanzania, Las Colorado in Mexico etc. are another examples of pink water bodies.
So Pink color of Lonar lake is because of change in microbial activity.

The low water level may result in increased salinity and changes in microbes behavior due to atmospheric changes, this may be the explanation for a change in color.