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.

Reference: https://pubmed.ncbi.nlm.nih.gov/32148310/

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.

Reference: https://www.biorxiv.org/content/10.1101/2020.07.03.186106v1

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.

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204287/

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.

Genomic analysis of a bacterial strain isolated from the Lonar Lake

-By Kranti Karande

Bacterial isolates of the Rhodococcus genus are widely recognized as being capable of catabolizing a wide variety of aliphatic, aromatic and polyaromatic hydrocarbons. Taxonomically genus Rhodococcus belongs to the Nocardiaceae family under phylum Actinobacteria. The tremendous catabolic diversity and remarkable biotransformation ability of genus Rhodococcus is mainly due to the existence of a diverse array of catabolic genes reported for several enzymatic groups such as monooxygenase, dioxygenase, and hydroxylases.

This study identified the genome sequencing data and the draft genome of the Rhodococcus rhodochrous strain originally isolated from Lonar Lake sediments located in Maharashtra province of India. The strain was identified as Rhodococcus rhodochrous based on the mass spectra of the cell extracts of overnight grown pure cultures and DNA sequencing results.

Specific gene and gene groups responsible for catabolism of various compounds have been identified and assigned to the respective compound. Total 39 genes were assigned to subsystem category ‘Stress response’ and the analysis of the genes for this subsystem category revealed the presence of specific genes. The presence of these specific genes implied that the isolate has the ability to synthesize and uptake ‘Glycine betaine (N,N,N-trimethylglycine)’ which may be the main osmolyte synthesized by this bacterium under osmotic stress conditions usually present in the soda lake environment. 

The genomic data reported in this publication will pave the way for further study of the different catabolic genes implicated in hydrocarbon metabolism in this bacterium.

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058525/

The microbial diversity of newly diagnosed diabetics is different from that of healthy non-diabetics, although there is no major difference in prediabetics.

– By Kranti Karande

Type 2 diabetes (T2D) is the most common type of diabetes. People who have T2D are said to have insulin resistance. T2D is a chronic condition that affects blood sugar (glucose) absorption and lipid metabolism.

Recently the gut microbiome was identified as an important factor for T2D development. Disruption of the balance between gut microbes has been linked to the development of metabolic diseases, in particular T2D, obesity and cardiovascular disorders. Some of the earlier studies observed dissimialrity between the gut microbiome of diabetics, prediabetics, and healthy nondiabetic individuals, although very few investigated the gut microbiome of treatment-naive individuals with T2D.

In this study, scientists have analyzed ND, PreDMs, NewDMs, and KnownDMs gut microbiome to understand and identify differences in the T2D, and prediabetes-associated microbial community. The scientists also looked at the community changes in microbial association networks and identified genera which are contributing for the transition from healthy (control) to diabetic state here called as driver taxa. They also analyzed the association of a wide array of serum biomarkers with genera, which were differentially abundant and were also found to be contributing for the major changes in the gut microbiome of T2D individuals .

In this study, a total of 102 subjects were studied, and they looked at the gut microbiota of prediabetics (PreDMs) (n= 17), newly diagnosed diabetics (NewDMs) (n = 11), and diabetics on antidiabetic treatment (KnownDMs) (n = 39) and compared them with healthy nondiabetics (ND) (n= 35). Twenty-five different serum biomarkers were measured to assess the status of diabetes and their association with gut microbiota.

The research identified nine separate genera in four sample groups as having differential abundance. Among them, Akkermansia, Blautia, and Ruminococcus were found to be significantly decreased, while Lactobacillus was increased in NewDMs compared to ND and recovered in KnownDMs. Akkermansia was inversely correlated with HbA1c and positively correlated with total antioxidants. Compared to ND, there was increased abundance of Megasphaera, Escherichia, and Acidaminococcus and decreased abundance of Sutterella in KnownDMs.

Among many taxa known to act as community drivers during disease progression, it was observed that genus Sutterella is a common driver taxon among all diabetic groups. On the basis of the results of random forest analysis (methodology) , they discovered that the serum metabolites fasting glucose, HbA1c, methionine, and total antioxidants were highly discriminating factors among studied groups. The compiled data showed that the gut microbial diversity of NewDMs is substantially different from that of ND but not of PreDMs. Interestingly, after anti-diabetic treatment, the microbial diversity of KnownDMs tends to recover toward that of ND.

Gut microbiota is thought to play a role in the development of the disease, and previous studies have documented a microbiome dysbiosis association with T2D. In this study, scientists have attempted to investigate gut microbiota of ND, PreDMs, NewDMs, and KnownDMs. They found that the genera Akkermansia and Blautia decreased significantly in treatment-naive diabetics and were restored in KnownDMs on antidiabetic treatment. Understanding the transition of microbiota and its association with serum biomarkers in diabetics with different disease states may pave the way for new therapeutic approaches for T2D.

Twenty-five different serum biomarkers were checked and compared with the gut microbiota to assess the different states of diabetes. Targeted 16S rRNA amplicon sequencing was used to assess the microbial diversity, community shuffling, and identification of driver taxa for the disease state. They have investigated relationships between a wide array of serum biomarkers responsible for progression of T2D with significantly diverged and differentially abundant taxa in each study group. Significantly different patterns were observed in the gut microbiota of PreDMs, NewDMs, and KnownDMs compared to ND. In KnownDMs, abundance of some microbial taxa was found to be similar to that of ND group.

Since oxidative stress is known to be involved in the establishment of insulin resistance and diabetic complications , they also measured total antioxidant capacity and lipid peroxides, a marker of oxidative damage to lipids in the blood. Interstingly, they found a significant decrease in total antioxidant capacity and increase in lipid peroxidation in treatment-naive NewDMs but not in PreDMs. In KnownDMs on treatment with metformin, an increase in total antioxidant capacity and decrease in lipid peroxidation were observed.

These findings show differences in the gut microbiome in PreDMs, NewDMs, and KnownDMs compared to ND. In PreDMs, the gut microbiota doesn’t show a significant difference when compared with ND, while in NewDMs, both abundance and diversity have changed significantly, which seems to be restored to some degree in KnownDMs on antidiabetic care.

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112960/pdf/mSystems.00578-19.pdf

Services at NCMR Pune

PART-II Microbial identification and characterization services

NCMR Pune offers multiple services for microbial identification and characterization. All of these services are listed below:

A. rRNA gene/ ITS sequencing for microbial identification: rRNA gene sequence of bacteria/ Internal Transcribed Sequence (ITS) of fungi is one of the most reliable molecular methods for microbial identification. We offer this sequencing service for identifying bacteria, archaea and fungi using universal primers for each type of organism.
Once the organism is received at NCMR, it goes through the following procedure: DNA isolation, PCR using universal primers for the type of organism, purification of the PCR amplicons, cycle sequencing reactions, purification and run them on an automated capillary-based Sanger DNA Sequencing system. At every step, there is in-house quality check to ensure success of the sequencing reactions. Post sequencing, fragments are manually checked and only good quality sequences are used to form contigs, which are then matched in well-curated databases for assigning closest neighbor as the tentative identification of your submitted organism.

B. Phylogenetic analysis: Phylogenetic analysis based on 16S rRNA gene is a powerful tool to study the evolutionary relationships among micro-organisms. It provides insights of genes or sequences which include relationships, origins, and closest taxonomic identification. These relationships are described by a branching of the tree.

C. Determination of Polar lipids: Lipids are one of the most verdiagram, or tree, with branches joined by nodes and leading to the terminal at the tips of satile compounds present in living system. Bacterial cell membrane is mostly composed of polar lipids like phospholipids, amino lipids and glycolipids etc. Lipids profile of a bacterial species is well suited to be used as a taxonomic character and is required to be included for description of novel taxa.

D. Determination of G+C mol% in DNA: G+C content in DNA of a species of bacteria is a characteristic feature and in bacterial world G+C mol% varies from 24% to 76%. This value is essential for description of a novel species.
There is a linear correlation between the melting temperature of DNA and G+C mol %. NCMR uses fluorimeteric method using double strand specific fluorescent dye and real-time PCR thermocyclers for determination of melting temperature (Tm) and G+C mol % is calculated from the Tm. This service includes isolation of high quality DNA from microbial culture(s), melting temperature experiment (in triplicate) and calculation of the G+C mol% content.

E. DNA-DNA hybridization: DNA-DNA hybridization (DDH) values determine relatedness between bacteria and is one of the minimum criteria for the description of novel taxon when 16S rRNA gene sequence similarity is more than 97%. Since 1960s DDH values have been used for species delineation with 70% as a recommended cut-off. At high temperature DNA gets denatured and when it cooled down gradually, it starts reassociating with the complementary sequences and results into the formation of double stranded DNA again. But when the DNA of two organisms mixed together and denatured, while reassociation of these hybrid DNA the degree of binding is directly proportional to the sequence similarity between the two DNAs. This degree of binding is converted into the percentage hybridisation. NCMR uses fluorimeteric method for the calculation of DDH.
This service includes isolation of high quality DNA from related species, hybridisation experiment (in triplicate) and calculation of the DDH value.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterisation includes: Colony morphology (such as size, shape, colour, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterization includes: Colony morphology (such as size, shape, color, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.
G. Phenotypic characterization by API: The Analytical Profile Index (API) is a miniaturized panel of biochemical tests compiled for identification of groups of closely related bacteria. Different test panels are available in dehydrated forms which are then reconstituted at the time of use by addition of bacterial suspensions of desired cell density. After proper incubation, test results are scored (after 24 and some time 48 hr) as +/- to generate a seven-digit code (profile). Identity of the bacterium is then derived from the database with the relevant cumulative profile code book or software (apiweb™).
NCMR offers phenotypic characterization by four type of API methods using either one or a combination of strips depending on the users’ requirements.
API 20E: API 20 E is a standardized characterization/identification system for Enterobacteriaceae and other non-fastidious, Gram-negative rods. It consists of 20 microtubes containing dehydrated substrates.
API 20NE: A standardized system characterization/identification of non-fastidious, non-enteric Gram-negative rods (e.g. Pseudomonas, Acinetobacter, Flavobacterium, Moraxella, Vibrio, Aeromonas, etc.), combining 8 conventional and 12 assimilation tests.
API 50 CH: Used for characterization/identification Bacillus and related genera as well as gram-negative rods belonging to Enterobacteriaceae and Vibrionaceae families based on fermentation of 49 carbohydrates.
API ZYM is a semi-quantitative micro-method designed for detection of enzymatic activities.

For more details on these services visit: http://210.212.161.138/services

Services at NCMR Pune

PART-I

National Centre for Microbial Resource, Pune is India’s microbial culture collection centre recognized by World Federation of Culture Collections (WFCC). The main objective of NCMR is to provide authentic microbial cultures to industries as well as academic and research institutes.

Culture collections play an important role in the area of biotechnology and are valuable resources for the sustainable use of microbial diversity and its conservation. But their meaningful exploitation is possible only if the properties of cultures are properly documented and the information is easily accessible. National Centre for Microbial Resource at NCCS Pune established by the Department of Biotechnology, Government of India is one among country’s largest culture collection centres. It is recognized as an International Depository Authority for the deposition of patent cultures under Budapest Treaty and designated national repository under the Biodiversity Act 2002.

NCMR focuses on fundamental research in the fields of microbial diversity, microbial taxonomy, microbial genomics and proteomics etc. One of the main objective of NCMR is providing microbial culture related services. NCMR provides multiple services like culture deposit, culture supply, identification/characterization of microorganisms, genomics/ metagenomics, imaging by scanning electron microscopy, phytoplasma detection, testing for microbial load for food safety certifications etc.

At present, NCMR accepts bacteria, fungi, and plasmids belonging to hazard group 1 and 2. Culture deposition service at NCMR is divided into following categories:

General deposit, IDA deposit and safe deposit.


A. General deposit: Cultures deposited under this category are accessible to public for teaching and /or scientific research and no restriction is imposed. NCMR reserves the right to reject a request for deposit of culture. Unidentified cultures are not accepted for deposit under this category. Once accepted and accessioned by NCMR, a culture cannot be withdrawn from the collection. There is no fee for such deposits.

B. International Depository Authority (IDA): Microorganisms deposited under IDA fulfill the requirement of the deposit for the purposes of patent procedures in all states signatory to the Budapest Treaty. NCMR is the second IDA in India. All IDAs operate as per the regulations of the Budapest treaty. The advantage of depositing microorganisms under IDA is almost similar forms are used and uniform procedures are followed in dealing with such deposits. Confidentiality and security of such deposits are maintained by NCMR. NCMR is obliged to keep the deposits under IDA for the period of 30 years from the date of deposit.

C. Safe deposit: Confidentiality and security is maintained while depositing such cultures. These cultures are not listed in the catalogue. There is an annual fee for such deposits. Depositor needs to sign an agreement with NCMR for making such a deposit.

NCMR supplies cultures listed in NCMR catalog (hard copy/electronic) for teaching and research purposes.

For more information on culture deposit and culture supply services at NCMR, visit following website: http://210.212.161.138/services

In the next article, we will learn about other services offered at NCMR.

Pea plant shapes its rhizosphere microbiome for nutrient uptake and combating stress

-By Kranti Karande

Legume crops like Pea are used as rotation crops along with rice cultivation in long term conservation agriculture experiments in the acidic soils of the North East region of India. Rhizosphere microbiomes present in the soil have significant influence on plant growth and productivity. The study aims at understanding the bacterial composition of microbiomes present in bulk soil as compared to the rhizosphere. It also aims to understand how the pea plant influences the bacterial communities present in soil and the rhizosphere microbiome in order to improve nutrient uptake and stress improvement. Pea cultivation is a practice used in conservation agriculture which strives to preserve and enrich the environmental resources to sustain and improve crop productivity. The study conducted will help devise future strategies to expand pea cultivation and improve soil health in the region. 

Crop rotation is an important and effective strategy as part of conservation agriculture practices. The North East region of India is a fragile, marginal, inaccessible and diverse ecosystem. Generally a mono-cropping system of rice is followed in this region.  Zero tillage (without disturbing the soil) cultivation of pea (Pisum sativum L.) has been considered beneficial to enhance the cropping intensity in the region. The majority of soils in North-East India are acidic in nature. The pH of soil among many other environmental factors has a significant influence on the type of nutrients and microorganisms present in the soil which in turn have an influence on the productivity of crops. Similarly, nutrient and residue management practices like the application of chemical fertilizers often influence the endogenous microbial communities. 

Sample collection for the study was done from experimental fields of the ICAR Research Complex for NEH Region, Umiam, Meghalaya, located in Eastern Himalayan region. Different tillage and residue management treatments were maintained in these fields for the last eight years by alternatively cultivating rice followed by pea cultivation. For microbial community analysis, bulk soil and pea rhizosphere samples were collected from each treatment plot. All the samples were processed for community DNA extraction. Analysis of the chemical properties of the soil samples was done using available methods. Rhizosphere soils were harvested from roots of pea plants. 

Soil pH (1:2.5) was found to be influenced by tillage and nutrient management practices at depth  0-15 cm. The combined effect of tillage and nutrient management practices on available N, P and K content and SOC,TOC of soil were significant. The rhizosphere showed higher diversity indices in comparison to the bulk soil samples. A total of 71 bacterial phyla were detected in the bulk soil and rhizosphere samples. A higher abundance of Firmicutes was recorded in bulk soil (~41.7%) in comparison to the rhizosphere (~17.8%). On the contrary, Proteobacteria were highly abundant in the rhizosphere (~43.9%) in comparison to bulk soil (~18.6%) samples. Significantly higher abundance of Proteobacteria and Bacteroidetes was observed in pea rhizosphere samples in comparison to bulk soil. 

Impact of residue management practices on abundance of specific microbial communities was observed across both rhizosphere and bulk soil samples. The impact of tillage history was also observed on the enrichment of specific OTUs in the bulk soil and rhizospheric soil. Differences in the abundance of 11 genera were recorded in the rhizosphere sample across the history of different tillage treatment. All these genera showed higher abundance in the conventional tillage fields. The correlation between soil properties and microbial community structure was also studied as part of the study. Significant correlations were observed between relative abundance of few bacterial phyla & genera and soil properties in both bulk soil and rhizospheric soil samples. However, the number of significant correlations was low in rhizosphere samples, in comparison to bulk soil samples. 

The study was designed to investigate the effect of long-term exposure to various tillage and residue management practices on the bacterial community structures of the bulk soils and how pea plant (a rotation crop) shapes the rhizosphere communities. A higher species diversity and evenness was observed in rhizospheric samples. There was no significant difference in bacterial richness and evenness among different tillage and residue management treatments in both rhizospheric and bulk soil samples. This is an indication that the plant rhizosphere effect (a plant’s ability to alter microbial communities in rhizospheric soil) is the key driver of alpha diversity. Plants can alter the microbial communities by secreting a variety of nutrients and bioactive molecules into the rhizosphere. Enrichment of specific OYUs in the Pea rhizosphere were also confirmed which can be attributed to the selection pressure of the Pea root. The results of the Pea rhizosphere and bulk soils were consistent with the fact that the majority of members of microbial communities in the host plant are horizontally acquired from the surrounding environment, and the soil is the main reservoir of a plant rhizosphere microbiome. The genus Nitrobacter was at higher abundance in pea rhizosphere samples than bulk soils, suggesting its enrichment by the host plant as Nitrobacter converts nitrite to nitrate making nitrogen more readily available to the host plant. Higher abundance of genes related to nitrogen fixation, phytohormone and siderophore production, phosphate solubilization in the rhizosphere soil substantiate the conclusion that the selection of bacterial communities is always based on plant growth promoting potential in the rhizosphere. 

The study concluded that pea plant is the most dominating selection factor shaping the microbial communities under diverse residue management and tillage treatments. The rhizospheric soil was found to be enriched with bacterial taxa known for plant growth promotion which indicates that the plant plays a role in selecting the rhizospheric communities to meet its requirement of nutrient uptake and combating stress.

Reference:
https://www.readcube.com/articles/10.3389/fmicb.2020.00968

Meet Dr. Om Prakash Sharma: Microbiologist at NCMR-NCCS Pune

You must have heard that ‘Oxygen is essential for Life!’ But what if I tell you that some organisms hate oxygen, and they cannot survive in presence of oxygen? Surprising! Isn’t it? There is a group of microorganisms called anaerobes who does not require oxygen for their survival. Dr. Om Prakash Sharma’s group at NCMR-NCCS Pune is interested in studying fascinating area of anaerobic microbiology. Dr. Sharma’s research interests also include environmental microbiology, microbial physiology, taxonomy etc. It was a great pleasure to interact with Dr. Sharma and know more about him as a person and as a researcher.

Kranti: What makes you most excited about working with anaerobes?
Dr. Sharma: Anaerobes are the provider of most of the anaerobic services like key components of clean-energy, global climate change, waste to energy generation, solid waste management, waste water treatment, bio-toilets, Human-gut-microbiome, anaerobic probiotics, fecal-microbiota-transplantation (FMT), fecal microbiome banking and emerging threat of anaerobic infection. However, they are less explored in comparison to aerobes as they are tough to cultivate and difficult to preserve. In India very few laboratories are working with anaerobes. I feel excited and energetic to think about the development of the biobank/ seedbank of obligate anaerobes and archaea  contributing to Indian academia and industries. This feeling pushes me to work with this group of microorganisms.

Kranti: How has your journey been from being a PhD student at Delhi University, Post-doctoral researcher at Florida State University, Georgia Institute of Technology to a Scientist at NCMR- NCCS Pune? How does research life change from being a student to being a faculty?
Dr. Sharma: My journey from DU to NCMR-NCCS Pune is same in terms of my feeling for microbes. I still feel like a student and have the same enthusiasm to explore the microbes. The best thing I feel is that I haven’t changed my field from last 20 years and it is helping me a lot to understand the field easily.  In terms of change, I am everyday learning about the feelings of students, colleagues and collaborators and try my level best not to hurt anyone in terms of ethics and benefit sharing. My 5 years’ postdoctoral experience was very fair about whom to give credit for what without hurting others and I try to implement that with my group too.

Kranti: We all are inspired or influenced by someone or something in life. Who or what is your inspiration in life?
Dr. Sharma: According to Hindu philosophy anyone who teaches you anything is a Guru and inspires your attitude and activities up to some extent. Although I appreciate the contribution of all my teachers but the work ethics, honesty and attitude of my postdoc advisor influenced me a lot. During my 4-5 years of postdoc duration, I always found him happy, smiling, well behaved, caring and learnt how to teach and appreciate your peers and subordinate without hurting or demoralizing  them.

Kranti: You grew up in a rural household, has it ever been a problem to achieve your dreams of becoming a scientist? 
Dr. Sharma: I can say yes in terms of money sometimes for essential books and application purposes but internally I never felt it as a problem. Fortunately, I got very good support from my teachers at each and every step of life that made me strong internally.

Kranti:  You are mentoring students for 10 years now. Every mentor has a unique way of training students. How do you train students? What is the most challenging part of training students?
Dr. Sharma: Till date I have only mentored students for their short period of dissertation but this number is quite good (40) and gives me the view of feeling of students. I would like to say getting good student is fortunate part of mentoring. During last 10 years some students were so nice in terms of knowledge and attitude that I realized that I was lucky to work with them. In addition, I never feel that I am mentor and more knowledgeable. I too learn from their observation, enthusiasm and way of working and writing and try to share what I have learnt.

Kranti: The major part of your research is focused on identification of Clostridium species, anaerobic infections, testing antimicrobial resistance and antimicrobial susceptibility of anaerobes. Could you elaborate on this part?
Dr. Sharma: I want to develop the culture bank of Clostridium of ecological and clinical significance and working on characterization of Clostridia available at NCMR . Anaerobic infection in humans is of developing interest among the clinicians and dentists and how the antibiotics behave in anaerobic vs anaerobic conditions have its own interest but availability of obligate anaerobes for all these purposes is of utmost importance. Cultivation and preservation of obligate anaerobes is a major challenge. Therefore according to mandate of NCMR we are reviving and developing long term preservation protocols that maintain the viability and functionality of the organisms for this purpose.

Kranti: Which methods and tools do you use in your research?
Dr. Sharma: It is really a very good question. Collecting anaerobic sample for cultivation of obligate anaerobic bacteria and Archaea is challenging. We use Hungate method of pre-reduced media preparation and handling. But for purification and cultivation of strict anaerobes availability of anoxic chamber and knowledge nutritional requirement, physiological nature and redox condition is a must. Considering the need of researchers working with strict anaerobes, we recently published a book Entitled Anaerobes and anaerobic process . Details of handling and techniques are given in that.

Kranti: You have received many recognition’s in your research career. Could you share your memory about any of the recognition?
Dr. Sharma: Till date I have received young achiever award from BHU, Best mentor Award from FAMU-USA, INSA-Visiting Fellow in Microbial Ecology, ICMR-DHR fellowshi in foreign laboratory and Members of Subcommittee of Methanogenic Archaea of ICSP. I was most thrilled during my visit to Israel as an INSA-Visiting Fellow in Microbial Ecology, my host prof. Ediie Cynrrun and myself worked together on same bench and I was influenced by him and learned how to treat and respect our guest when anyone visit our lab or facility.

Kranti:  Could you shed some light on where India stands today in Anaerobic microbiology research?
Dr. Sharma: Due to time consuming nature of handling of strict anaerobes very few researchers and student are inclined towards anaerobes. Most of the culture coming from Indian laboratories for deposit purpose on name of anaerobes are either facultative or aerotolerants. Due to increasing importance of anaerobes in gut research and biogas, Indian researchers are focusing on cultivation, characterization and biobanking.

Kranti: How do you maintain the balance of your family life and work life?
Dr. Sharma : It is a very difficult question to answer. I am waiting for my  kids to grow.

Kranti: Would you share with us any memorable incident/moment of your research life?
Dr. Sharma: I always have a fascination to work on weekends for half a day. Once I was working in Florida State University alone on Saturday and melting the agar-medium inside the tightly closed serum vials. When I picked the vials in hand from the water bath it blasted like a bomb. Fortunately, I was following the safety protocols and wore PPE. After that I immediately came home and never planned a risky experiment on the weekend when I am alone.

Kranti: Does science become hectic sometimes? What do you do to relax?
Dr. Sharma: Yes; It is  very natural. When I feel saturated I watch old movies, listen to gazals, write some poems and read literature.

Kranti: Are journals necessary in the age of the internet? Don’t you think research should be done not just to publish a paper but also to have real life impacts? 
Dr. Sharma: Publications of finding are an integral part of research but it depends what you are publishing and what is your ultimate aim and how your peers evaluate you. Number of publications depends on the type of work. But I agree research should not be done only for publication.

Kranti: Being a rational person, what do you think about the state of scientific temperament in the current times? 
Dr. Sharma: Scientific temperament is an individual property but it also very much depends on institute, laboratory environment, group leaders, individual interest, scientific ethics and honesty.

Kranti: What advice would you give to the young generation who want to pursue research?
Dr. Sharma: In my view honesty, ethics and interest matters for science and if anyone has all the three he/she will definitely enjoy it.

Kranti: How will you brief about your research if you want to communicate it to a layman?
Dr.Sharma: I am working with tiny unseen creatures of life. We cultivate, nourish and preserve them. They do not need oxygen for survival.  They, themselves, reside in dark and extreme pain but give light and energy to others. They are responsible for global climate change and global warming. Generate waste to energy, produce biogas and bio-fertilizer from waste. Essential component of gut-heath, bio-toilets and sewage treatment plant but also responsible for infection of deeper part of human-body.

Kranti: Where do you think India stands today in science communication? How can scientists contribute to effective science communication?
Dr. Sharma: The ultimate aim of science is to serve society. It will be only possible with more communication. We need more work in this area. Spreading science to the layman in popular form is also the responsibility of scientists. In my view it can be in any form cartoon, model, poems, scientific talks and popular writing etc. It should be promoted by funding agencies as well as at institutional and personal laboratory levels. Communication will also incline new generation scientists for doing better science.