Anaerobic biodegradation of phenanthrene and pyrene by sulfate-reducing cultures enriched from contaminated freshwater lake sediments.

Our current understanding of the susceptibility of hazardous polycyclic aromatic hydrocarbons (PAHs) to anaerobic microbial degradation is very limited. In the present study, we obtained phenanthrene- and pyrene-degrading strictly anaerobic sulfate-reducing enrichments using contaminated freshwater lake sediments as the source material. The highly enriched phenanthrene-degrading culture, MMKS23, was dominated (98%) by a sulfate-reducing bacterium belonging to the genus Desulfovibrio. While Desulfovibrio sp. was also predominant (79%) in the pyrene-degrading enrichment culture, MMKS44, an anoxygenic purple non-sulfur bacterium, Rhodopseudomonas sp., constituted a significant fraction (18%) of the total microbial community. Phenanthrene or pyrene biodegradation by the enrichment cultures was coupled with sulfate reduction, as evident from near stoichiometric consumption of sulfate and accumulation of sulfide. Also, there was almost complete inhibition of substrate degradation in the presence of an inhibitor of sulfate reduction, i.e., 20 mM MoO42-, in the culture medium. After 180 days of incubation, about 79.40 µM phenanthrene was degraded in the MMKS23 culture, resulting in the consumption of 806.80 µM sulfate and accumulation of 625.80 µM sulfide. Anaerobic pyrene biodegradation by the MMKS44 culture was relatively slow. About 22.30 µM of the substrate was degraded after 180 days resulting in the depletion of 239 µM sulfate and accumulation of 196.90 µM sulfide. Biodegradation of phenanthrene by the enrichment yielded a metabolite, phenanthrene-2-carboxylic acid, suggesting that carboxylation could be a widespread initial step of phenanthrene activation under sulfate-reducing conditions. Overall, this novel study demonstrates the ability of sulfate-reducing bacteria (SRB), dwelling in contaminated freshwater sediments to anaerobically biodegrade three-ringed phenanthrene and highly recalcitrant four-ringed pyrene. Our findings suggest that SRB could play a crucial role in the natural attenuation of PAHs in anoxic freshwater sediments.

Impact of Nitrate and Ammonium Concentrations on Co-Culturing of Tetradesmus obliquus IS2 with Variovorax paradoxus IS1 as Revealed by Phenotypic Responses.

Mutual interactions in co-cultures of microalgae and bacteria are well known for establishing consortia and nutrient uptake in aquatic habitats, but the phenotypic changes in terms of morphological, physiological, and biochemical attributes that drive these interactions have not been clearly understood. In this novel study, we demonstrated the phenotypic response in a co-culture involving a microalga, Tetradesmus obliquus IS2, and a bacterium, Variovorax paradoxus IS1, grown with varying concentrations of two inorganic nitrogen sources. Modified Bold's basal medium was supplemented with five ratios (%) of NO3-N:NH4-N (100:0, 75:25, 50:50, 25:75, and 0:100), and by maintaining N:P Redfield ratio of 16:1. The observed morphological changes in microalga included an increase in granularity and a broad range of cell sizes under the influence of increased ammonium levels. Co-culturing in presence of NO3-N alone or combination with NH4-N up to equimolar concentrations resulted in complete nitrogen uptake, increased growth in both the microbial strains, and enhanced accumulation of carbohydrates, proteins, and lipids. Total chlorophyll content in microalga was also significantly higher when it was grown as a co-culture with NO3-N and NH4-N up to a ratio of 50:50. Significant upregulation in the synthesis of amino acids and sugars and downregulation of organic acids were evident with higher ammonium uptake in the co-culture, indicating the regulation of carbon and nitrogen assimilation pathways and energy synthesis. Our data suggest that the co-culture of strains IS1 and IS2 could be exploited for effluent treatment by considering the concentrations of inorganic sources, particularly ammonium, in the wastewaters.

Extracellular Polymeric Substances Drive Symbiotic Interactions in Bacterial‒Microalgal Consortia.

The importance of several factors that drive the symbiotic interactions between bacteria and microalgae in consortia has been well realised. However, the implication of extracellular polymeric substances (EPS) released by the partners remains unclear. Therefore, the present study focused on the influence of EPS in developing consortia of a bacterium, Variovorax paradoxus IS1, with a microalga, Tetradesmus obliquus IS2 or Coelastrella sp. IS3, all isolated from poultry slaughterhouse wastewater. The bacterium increased the specific growth rates of microalgal species significantly in the consortia by enhancing the uptake of nitrate (88‒99%) and phosphate (92‒95%) besides accumulating higher amounts of carbohydrates and proteins. The EPS obtained from exudates, collected from the bacterial or microalgal cultures, contained numerous phytohormones, vitamins, polysaccharides and amino acids that are likely involved in interspecies interactions. The addition of EPS obtained from V. paradoxus IS1 to the culture medium doubled the growth of both the microalgal strains. The EPS collected from T. obliquus IS2 significantly increased the growth of V. paradoxus IS1, but there was no apparent change in bacterial growth when it was cultured in the presence of EPS from Coelastrella sp. IS3. These observations indicate that the interaction between V. paradoxus IS1 and T. obliquus IS2 was mutualism, while commensalism was the interaction between the bacterial strain and Coelastrella sp. IS3. Our present findings thus, for the first time, unveil the EPS-induced symbiotic interactions among the partners involved in bacterial‒microalgal consortia.

Sustainability Evaluation of Immobilized Acid-Adapted Microalgal Technology in Acid Mine Drainage Remediation following Emergy and Carbon Footprint Analysis.

Sustainability evaluation of wastewater treatment helps to reduce greenhouse gas emissions, as it emphasizes the development of green technologies and optimum resource use rather than the end-of-pipe treatment. The conventional approaches for treating acid mine drainages (AMDs) are efficient; however, they need enormous amounts of energy, making them less sustainable and causing greater environmental concern. We recently demonstrated the potential of immobilized acid-adapted microalgal technology for AMD remediation. Here, this novel approach has been evaluated following emergy and carbon footprint analysis for its sustainability in AMD treatment. Our results showed that imported energy inputs contributed significantly (>90%) to the overall emergy and were much lower than in passive and active treatment systems. The microalgal treatment required 2-15 times more renewable inputs than the other two treatment systems. Additionally, the emergy indices indicated higher environmental loading ratio and lower per cent renewability, suggesting the need for adequate renewable inputs in the immobilized microalgal system. The emergy yield ratio for biodiesel production from the microalgal biomass after AMD treatment was >1.0, which indicates a better emergy return on total emergy spent. Based on greenhouse gas emissions, carbon footprint analysis (CFA), was performed using default emission factors, in accordance with the IPCC standards and the National Greenhouse Energy Reporting (NGER) program of Australia. Interestingly, CFA of acid-adapted microalgal technology revealed significant greenhouse gas emissions due to usage of various construction materials as per IPCC, while SCOPE 2 emissions from purchased electricity were evident as per NGER. Our findings indicate that the immobilized microalgal technology is highly sustainable in AMD treatment, and its potential could be realized further by including solar energy into the overall treatment system.

Cyto-genotoxicity evaluation of pyroligneous acid using Allium cepa assay.

Pyroligneous acid (PA) is a highly oxygenated organic condensate obtained by cooling the gases generated from the pyrolysis process. PA has been used in agriculture for several years with multiple beneficial effects, including plant health and yields, pest resilience, and seed germination. It is generally applied to agricultural soils in the dilution of 1:1000 to 1:100, corresponding to 0.1-1% PA concentration. In this study, the cyto-genotoxic potential of PA to Allium cepa meristematic root-tips (where all cells undergo repeated division and form primary root tissues) was examined. Exposure to PA concentrations of 0.1% and above showed a reduction in the mitotic index percentage, and at 5%, a complete arrest in the cell division was recorded. However, chromosomal aberrations at 0.5, 1, and 3% PA were reversible types such as bridges, vagrants, laggards, and multipolar anaphase, with a maximum of only 5.8% chromosomal aberration observed at 3% PA. Comet assay (single-cell gel electrophoresis) for genotoxicity assessment determined using PA exposed A. cepa root tips showed that it was not genotoxic. The absence of cyto-genotoxicity in A. cepa, even at concentrations far above what would be typically encountered in agricultural applications, strongly suggests that PA is unlikely to cause adverse effects on crops and ultimately on the biota and human health.

Soil microalgae and cyanobacteria: the biotechnological potential in the maintenance of soil fertility and health.

The soil microbiota plays a major role in maintaining the nutrient balance, carbon sink, and soil health. Numerous studies reported on the function of microbiota such as plant growth-promoting bacteria and fungi in soil. Although microalgae and cyanobacteria are ubiquitous in soil, very less attention has been paid on the potential of these microorganisms. The indiscriminate use of various chemicals to enhance agricultural productivity led to serious consequences like structure instability, accumulation of toxic contaminants, etc., leading to an ecological imbalance between soil, plant, and microbiota. However, the significant role of microalgae and cyanobacteria in crop productivity and other potential options has been so far undermined. The intent of the present critical review is to highlight the significance of this unique group of microorganisms in terms of maintaining soil fertility and soil health. Beneficial soil ecological applications of these two groups in enhancing plant growth, establishing interrelationships among other microbes, and detoxifying chemical agents such as insecticides, herbicides, etc. through mutualistic cooperation by synthesizing enzymes and phytohormones are presented. Since recombinant technology involving genomic integration favors the development of useful traits in microalgae and cyanobacteria for their potential application in improvement of soil fertility and health, the merits and demerits of various such advanced methodologies associated in harnessing the biotechnological potential of these photosynthetic microorganisms for sustainable agriculture were also discussed.

Advances in the technologies for studying consortia of bacteria and cyanobacteria/microalgae in wastewaters.

The excessive generation and discharge of wastewaters have been serious concerns worldwide in the recent past. From an environmental friendly perspective, bacteria, cyanobacteria and microalgae, and the consortia have been largely considered for biological treatment of wastewaters. For efficient use of bacteria‒cyanobacteria/microalgae consortia in wastewater treatment, detailed knowledge on their structure, behavior and interaction is essential. In this direction, specific analytical tools and techniques play a significant role in studying these consortia. This review presents a critical perspective on physical, biochemical and molecular techniques such as microscopy, flow cytometry with cell sorting, nanoSIMS and omics approaches used for systematic investigations of the structure and function, particularly nutrient removal potential of bacteria‒cyanobacteria/microalgae consortia. In particular, the use of specific molecular techniques of genomics, transcriptomics, proteomics metabolomics and genetic engineering to develop more stable consortia of bacteria and cyanobacteria/microalgae with their improved biotechnological capabilities in wastewater treatment has been highlighted.

Nutrient removal and biomass production: advances in microalgal biotechnology for wastewater treatment.

Owing to certain drawbacks, such as energy-intensive operations in conventional modes of wastewater treatment (WWT), there has been an extensive search for alternative strategies in treatment technology. Biological modes for treating wastewaters are one of the finest technologies in terms of economy and efficiency. An integrated biological approach with chemical flocculation is being conventionally practiced in several-sewage and effluent treatment plants around the world. Overwhelming responsiveness to treat wastewaters especially by using microalgae is due to their simplest photosynthetic mechanism and ease of acclimation to various habitats. Microalgal technology, also known as phycoremediation, has been in use for WWT since 1950s. Various strategies for the cultivation of microalgae in WWT systems are evolving faster. However, the availability of innovative approaches for maximizing the treatment efficiency, coupled with biomass productivity, remains the major bottleneck for commercialization of microalgal technology. Investment costs and invasive parameters also delimit the use of microalgae in WWT. This review critically discusses the merits and demerits of microalgal cultivation strategies recently developed for maximum pollutant removal as well as biomass productivity. Also, the potential of algal biofilm technology in pollutant removal, and harvesting the microalgal biomass using different techniques have been highlighted. Finally, an economic assessment of the currently available methods has been made to validate microalgal cultivation in wastewater at the commercial level.

Microalgae-bacteria biofilms: a sustainable synergistic approach in remediation of acid mine drainage.

Microalgae and bacteria offer a huge potential in delving interest to study and explore various mechanisms under extreme environments. Acid mine drainage (AMD) is one such environment which is extremely acidic containing copious amounts of heavy metals and poses a major threat to the ecosystem. Despite its extreme conditions, AMD is the habitat for several microbes and their activities. The use of various chemicals in prevention of AMD formation and conventional treatment in a larger scale is not feasible under different geological conditions. It implies that microbe-mediated approach is a viable and sustainable alternative technology for AMD remediation. Microalgae in biofilms play a pivotal role in such bioremediation as they maintain mutualism with heterotrophic bacteria. Synergistic approach of using microalgae-bacteria biofilms provides supportive metabolites from algal biomass for growth of bacteria and mediates remediation of AMD. However, by virtue of their physiology and capabilities of metal removal, non-acidophilic microalgae can be acclimated for use in AMD remediation. A combination of selective acidophilic and non-acidophilic microalgae together with bacteria, all in the form of biofilms, may be very effective for bioremediation of metal-contaminated waters. The present review critically examines the nature of mutualistic interactions established between microalgae and bacteria in biofilms and their role in removal of metals from AMDs, and consequent biomass production for the yield of biofuel. Integration of microalgal-bacterial consortia in fuel cells would be an attractive emerging approach of microbial biotechnology for AMD remediation.

`A critical review on current status and environmental sustainability of pre-treatment methods for bioethanol production from lignocellulose feedstocks.

Lignocellulosic biomass resource has been widely used as a natural resource for the synthesis of biofuels and bio-based products through pre-treatment, saccharification and fermentation processes. In this review, we delve into the environmental implications of bioethanol production from the widely utilized lignocellulosic biomass resource. The focus of our study is the critical stage of pre-treatment in the synthesis process, which also includes saccharification and fermentation. By collecting scientific data from the available literature, we conducted a comprehensive life cycle analysis. Our findings revealed substantial differences in the environmental burdens associated with diverse pre-treatment methods used for lignocellulosic biomass. These results highlight the importance of selecting environmentally benign pre-treatment techniques to promote the sustainability of bioethanol production. Future research directions are suggested, emphasizing the optimization of pre-treatment processes to further mitigate their environmental impact.

Microwave-assisted alkali pre-treatment medium for fractionation of rice straw and catalytic conversion to value-added 5-hydroxymethyl furfural and lignin production.

In the current study, the use of microwave-assisted sodium hydroxide medium (MWSH) for pre-treatment and saccharification of rice straw to obtain sugar syrup for the production of 5-hydroxymethyl furfural (5-HMF) was investigated. The optimization of the MWSH pre-treatment was carried out using central composite methodology, resulting in a maximum reducing sugar yield of 350 mg/g of treated rice straw (TRS) and a glucose yield of 255 mg/g of TRS under the conditions of a microwave power of 681 W, a NaOH concentration of 0.54 M, and a pre-treatment time of 3 min. Additionally, the microwave assisted transformation of sugar syrup with titanium magnetic silica nanoparticle as catalyst, producing 41.1 % yield of 5-HMF from the sugar syrup after 30 min microwave irradiation at 120 °C with catalyst loading of 2.0:200 (w/v)). The structural characterization of the lignin was analysed using 1H NMR techniques, and the surface carbon (C1s spectra) and oxygen (O1s spectra) composition changes of the rice straw during pre-treatment were analysed using X-ray photoelectron spectroscopy. The rice straw based bio-refinery process which contains MWSH pretreatment followed by dehydration of sugars achieved high efficiency of 5-HMF production.

Phenotypic changes in microalgae at acidic pH mediate their tolerance to higher concentrations of transition metals.

Acclimatory phenotypic response is a common phenomenon in microalgae, particularly during heavy metal stress. It is not clear so far whether acclimating to one abiotic stressor can alleviate the stress imposed by another abiotic factor. The intent of the present study was to demonstrate the implication of acidic pH in effecting phenotypic changes that facilitate microalgal tolerance to biologically excess concentrations of heavy metals. Two microalgal strains, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were exposed to biologically excess concentrations of Cu (0.50 and 1.0 mg L‒1), Fe (5 and 10 mg L‒1), Mn (5 and 10 mg L‒1) and Zn (2, 5 and 10 mg L‒1) supplemented to the culture medium at pH 3.5 and 6.7. Chlorophyll autofluorescence and biochemical fingerprinting using FTIR-spectroscopy were used to assess the microalgal strains for phenotypic changes that mediate tolerance to metals. Both the strains responded to acidic pH by effecting differential changes in biochemicals such as carbohydrates, proteins, and lipids. Both the microalgal strains, when acclimated to low pH of 3.5, exhibited an increase in protein (< 2-fold) and lipid (> 1.5-fold). Strain MAS1 grown at pH 3.5 showed a reduction (1.5-fold) in carbohydrates while strain MAS3 exhibited a 17-fold increase in carbohydrates as compared to their growth at pH 6.7. However, lower levels of biologically excess concentrations of the selected transition metals at pH 6.7 unveiled positive or no effect on physiology and biochemistry in microalgal strains, whereas growth with higher metal concentrations at this pH resulted in decreased chlorophyll content. Although the bioavailability of free-metal ions is higher at pH 3.5, as revealed by Visual MINTEQ model, no adverse effect was observed on chlorophyll content in cells grown at pH 3.5 than at pH 6.7. Furthermore, increasing concentrations of Fe, Mn and Zn significantly upregulated the carbohydrate metabolism, but not protein and lipid synthesis, in both strains at pH 3.5 as compared to their growth at pH 6.7. Overall, the impact of pH 3.5 on growth response suggested that acclimation of microalgal strains to acidic pH alleviates metal toxicity by triggering physiological and biochemical changes in microalgae for their survival.

Microalgal-bacterial consortia unveil distinct physiological changes to facilitate growth of microalgae.

Physiological changes that drive the microalgal-bacterial consortia are poorly understood so far. In the present novel study, we initially assessed five morphologically distinct microalgae for their ability in establishing consortia in Bold's basal medium with a bacterial strain, Variovorax paradoxus IS1, all isolated from wastewaters. Tetradesmus obliquus IS2 and Coelastrella sp. IS3 were further selected for gaining insights into physiological changes, including those of metabolomes in consortia involving V. paradoxus IS1. The distinct parameters investigated were pigments (chlorophyll a, b, and carotenoids), reactive oxygen species (ROS), lipids and metabolites that are implicated in major metabolic pathways. There was a significant increase (>1.2-fold) in pigments, viz., chlorophyll a, b and carotenoids, decrease in ROS and an enhanced lipid yield (>2-fold) in consortia than in individual cultures. In addition, the differential regulation of cellular metabolites such as sugars, amino acids, organic acids and phytohormones was distinct among the two microalgal-bacterial consortia. Our results thus indicate that the selected microalgal strains, T. obliquus IS2 and Coelastrella sp. IS3, developed efficient consortia with V. paradoxus IS1 by effecting the required physiological changes, including metabolomics. Such microalgal-bacterial consortia could largely be used in wastewater treatment and for production of value-added metabolites.

Sustainable Iron Recovery and Biodiesel Yield by Acid-Adapted Microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, Grown in Synthetic Acid Mine Drainage.

Sustainable resource recovery is the key to manage the overburden of various waste entities of mining practices. The present study demonstrates for the first time a novel approach for iron recovery and biodiesel yield from two acid-adapted microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, grown in synthetic acid mine drainage (SAMD). Virtually, there was no difference in the growth of the strain MAS3 both in Bold's basal medium (control) and SAMD. Using the IC50 level (200 mg L-1) and a lower concentration (50 mg L-1) of iron in SAMD, the cell granularity, exopolysaccharide (EPS) secretion, iron recovery, and biodiesel were assessed in both the strains. Both cell granularity and accumulation of EPS were significantly altered under metal stress in SAMD, resulting in an increase in total accumulation of iron. Growth of the microalgal strains in SAMD yielded 12-20% biodiesel, with no traces of heavy metals, from the biomass. The entire amount of iron, accumulated intracellularly, was recovered in the residual biomass. Our results on the ability of the acid-adapted microalgal strains in iron recovery and yield of biodiesel when grown in SAMD indicate that they could be the potential candidates for use in bioremediation of extreme habitats like AMD.

Acid-adapted microalgae exhibit phenotypic changes for their survival in acid mine drainage samples.

Phenotypic plasticity or genetic adaptation in an organism provides phenotypic changes when exposed to the extreme environmental conditions. The resultant physiological and metabolic changes greatly enhance the organism's potential for its survival in such harsh environments. In the present novel approach, we tested the hypothesis whether acid-adapted microalgae, initially isolated from non-acidophilic environments, can survive and grow in acid-mine-drainage (AMD) samples. Two acid-adapted microalgal strains, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were tested individually or in combination (co-culture) for phenotypic changes during their growth in samples collected from AMD. The acid-adapted microalgae in AMD exhibited a two-fold increase in growth when compared with those grown at pH 3.5 in BBM up to 48 h and then declined. Furthermore, oxidative stress triggered several alterations such as increased cell size, granularity, and enhanced lipid accumulation in AMD-grown microalgae. Especially, the apparent limitation of phosphate in AMD inhibited the uptake of copper and iron in the cultures. Interestingly, growth of the acid-adapted microalgae in AMD downregulated amino acid metabolic pathways as a survival mechanism. This study demonstrates for the first time that acid-adapted microalgae can survive under extreme environmental conditions as exist in AMD by effecting significant phenotypic changes.

Acid-tolerant microalgae can withstand higher concentrations of invasive cadmium and produce sustainable biomass and biodiesel at pH 3.5.

Two acid-tolerant microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, originally isolated from non-acidophilic environment, were tested for their ability to withstand higher concentrations of an invasive heavy metal, cadmium (Cd), at an acidic pH of 3.5 and produce biomass rich in biodiesel. The growth analysis, in terms of chlorophyll, revealed that strain MAS1 was tolerant even to 20 mg L-1 of Cd while strain MAS3 could withstand only up to 5 mg L-1. When grown in the presence of 2 mg L-1, a concentration which is 400-fold higher than that usually occurs in the environment, the microalgal strains accumulated >58% of Cd from culture medium at pH 3.5. FTIR analysis of Cd-laden biomass indicated production of significant amounts of biodiesel rich in fatty acid esters. This is the first study that demonstrates the capability of acid-tolerant microalgae to grow well and remove Cd at acidic pH.

Sustainable production of biomass and biodiesel by acclimation of non-acidophilic microalgae to acidic conditions.

The overwhelming response towards algal biodiesel production has been well-recognized recently as a sustainable alternative to conventional fuels. Most microalgae cannot grow well at acidic pH. The present study, therefore, investigated whether non-acidophilic microalgae Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3 can be acclimated to extreme-acidic pH for sustainable production of biomass and biodiesel. Growth analysis indicated that both the microalgal strains possessed a passive uptake of CO2 at pH 3.0 with biomass production of 0.25 g dry wt. L-1 in Desmodemus sp. and 0.45 g dry wt. L-1 in Heterochlorella sp.. Flow-cytometry analysis for reactive oxygen species, membrane permeability and neutral-lipids revealed the capabilities of both strains to adapt to the stress imposed by acidic pH. Lipid production was doubled in both the strains when grown at pH 3.0. In-situ transesterification of biomass resulted in 13-15% FAME yield in the selected microalgae, indicating their great potential in biofuel production.

Potential of acid-tolerant microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, in heavy metal removal and biodiesel production at acidic pH.

Metals in traces are vital for microalgae but their occurrence at high concentrations in habitats is a serious ecological concern. We investigated the potential of two acid-tolerant microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, isolated from neutral environments, for simultaneous removal of heavy metals such as copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn), and production of biodiesel when grown at pH 3.5. Excepting Cu, the selected metals at concentrations of 10-20 mg L-1 supported good growth of both the strains. Cellular analysis for metal removal revealed the predominance of intracellular mechanism in both the strains resulting in 40-80 and 40-60% removal of Fe and Mn, respectively. In-situ transesterification of biomass indicated enhanced biodiesel yield with increasing concentrations of metals suggesting that both these acid-tolerant microalgae may be the suitable candidates for simultaneous remediation, and sustainable biomass and biodiesel production in environments like metal-rich acid mine drainages.