Anaerobic Degradation of Naphthalene and Pyrene by Sulfate-Reducing Cultures Enriched from Former Manufactured Gas Plant Soil.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) under completely anaerobic sulfate-reducing conditions is an energetically challenging process. To date, anaerobic degradations of only two-ringed naphthalene and three-ringed phenanthrene by sediment-free and enriched sulfate-reducing bacteria have been reported. In this study, sulfate-reducing enrichment cultures capable of degrading naphthalene and four-ringed PAH, pyrene, were enriched from a contaminated former gas plant site soil. Bacterial community composition analysis revealed that a naphthalene-degrading enrichment culture, MMNap, was dominated (84.90%) by a Gram-positive endospore-forming member of the genus Desulfotomaculum with minor contribution (8.60%) from a member of Clostridium. The pyrene-degrading enrichment, MMPyr, was dominated (97.40%) by a species of Desulfotomaculum. The sequences representing the Desulfotomaculum phylotypes shared 98.80% similarity to each other. After 150 days of incubation, MMNap degraded 195 µM naphthalene with simultaneous reduction of sulfate and accumulation of sulfide. Similarly, MMPyr degraded 114 µM pyrene during 180 days of incubation with nearly stochiometric sulfate consumption and sulfide accumulation. In both cases, the addition of sulfate reduction inhibitor, molybdate (20 mM), resulted in complete cessation of the substrate utilization and sulfate reduction that clearly indicated the major role of the sulfate-reducing Desulfotomaculum in biodegradation of the two PAHs. This study is the first report on anaerobic pyrene degradation by a matrix-free, strictly anaerobic, and sulfate-reducing enrichment culture.

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.

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.

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.

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.

Anaerobic Microbial Degradation of Polycyclic Aromatic Hydrocarbons: A Comprehensive Review.

Polycyclic aromatic hydrocarbons (PAHs) are a class of hazardous organic contaminants that are widely distributed in nature, and many of them are potentially toxic to humans and other living organisms. Biodegradation is the major route of detoxification and removal of PAHs from the environment. Aerobic biodegradation of PAHs has been the subject of extensive research; however, reports on anaerobic biodegradation of PAHs are so far limited. Microbial degradation of PAHs under anaerobic conditions is difficult because of the slow growth rate of anaerobes and low energy yield in the metabolic processes. Despite the limitations, some anaerobic bacteria degrade PAHs under nitrate-reducing, sulfate-reducing, iron-reducing, and methanogenic conditions. Anaerobic biodegradation, though relatively slow, is a significant process of natural attenuation of PAHs from the impacted anoxic environments such as sediments, subsurface soils, and aquifers. This review is intended to provide comprehensive details on microbial degradation of PAHs under various reducing conditions, to describe the degradation mechanisms, and to identify the areas that should receive due attention in further investigations.

Endophyte-assisted phytoremediation: mechanisms and current application strategies for soil mixed pollutants.

Phytoremediation uses plants and associated microbes to remove pollutants from the environment and is considered a promising bioremediation method. Compared with well-described single contaminant treatments, the number of studies reporting phytoremediation of soil mixed pollutants has increased recently. Endophytes, including bacteria and fungi, exhibit beneficial traits for the promotion of plant growth, stress alleviation, and biodegradation. Moreover, endophytes either directly or indirectly assist host plants to survive high concentrations of organic and inorganic pollutants in the soil. Endophytic microorganisms can also regulate the plant metabolism in different ways, exhibiting a variety of physiological characteristics. This review summarizes the taxa and physiological properties of endophytic microorganisms that may participate in the detoxification of contaminant mixtures. Furthermore, potential biomolecules that may enhance endophyte mediated phytoremediation are discussed. The practical applications of pollutant-degrading endophytes and current strategies for applying this valuable bio-resource to soil phytoremediation are summarized.

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.

Bioremediation of soil long-term contaminated with PAHs by algal-bacterial synergy of Chlorella sp. MM3 and Rhodococcus wratislaviensis strain 9 in slurry phase.

Remediation of soil contaminated with pollutants using biological agents is more a sustainable and greener approach as compared to physico-chemical technologies. We recently confirmed that a microalga, Chlorella sp. MM3, and a bacterium, Rhodococcus wratislaviensis strain 9, can degrade high-molecular weight PAHs. In this study, an algal-bacterial system of these two strains was developed by long-term growth on a mixture of phenanthrene, pyrene, and benzo[a]pyrene (BaP). In a soil spiked with 50 mg L-1 phenanthrene, 10 mg L-1 of pyrene and 10 mg L-1 of BaP, the algal-bacterial system degraded these PAHs almost completely in slurry phase within 30 days. Also, the algal-bacterial system was able to successfully remediate these three PAHs in a soil long-term contaminated with 245.1 mg kg-1 of 16 PAHs and several heavy metals under slurry phase in 21 days. Use of such appropriate assays as chlorophyll estimation for the microalga and semi-quantitative PCR for the bacterium confirmed survival of both the strains during soil bioremediation. Moreover, the residual toxicity test involving Escherichia coli DH5α that expresses green fluorescent protein indicated the successful bioremediation of PAHs-contaminated soil in slurry phase. For the first time, here we demonstrate the great potential of an algal-bacterial synergy in bioremediation of soil long-term contaminated with PAHs even in the presence of toxic heavy metals.

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.

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.

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.

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.

Biodegradation of high-molecular weight PAHs by Rhodococcus wratislaviensis strain 9: Overexpression of amidohydrolase induced by pyrene and BaP.

A Gram-positive bacterium, Rhodococcus wratislaviensis strain 9, completely degraded 280 µM of phenanthrene, 40% of 50 µM pyrene or 28% of 40 µM benzo[a]pyrene (BaP), each supplemented in M9 medium, within 7 days. PCR screening with gene-specific primers indicated that the strain 9 harbors genes which code for 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC), 4-nitrophenol 2-monooxygenase component B (npcB) as well as oxygenase component (nphA1), 4-hydroxybenzoate 3-monooxygenase (phbH), extradiol dioxygenase (edo), and naphthalene dioxygenase (ndo), all of which are largely implicated in biodegradation of several aromatic hydrocarbons. An orthogonal design experiment revealed that BaP biodegradation was greatly enhanced by surfactants such as Tween 80, Triton X-100 and linoleic acid, suggesting that bioavailability is the major limiting factor in bacterial metabolism of BaP. Both pyrene and BaP induced the overexpression of amidohydrolase, a metallo-dependent hydrolase, possibly involved in their biodegradation by strain 9. The up-regulation of amidohydrolase gene induced by BaP, in particular, was also confirmed by semi-quantitative RT-PCR. Catechol 2,3-dioxygenase and the large subunit of ndo, but not amidohydrolase, accumulated when the strain 9 was grown on phenanthrene. To our knowledge, this is the first report on overexpression of amidohydrolase and its possible implication in bacterial degradation of high-molecular weight PAHs.

Draft Genome Sequence of Microbacterium esteraromaticum MM1, a Bacterium That Hydrolyzes the Organophosphorus Pesticide Fenamiphos, Isolated from Golf Course Soil.

In this study, we report the first draft genome sequence of Microbacterium esteraromaticum MM1, isolated from golf course soil in South Australia. The genome possesses genes for the hydrolysis of organophosphorus (OP) pesticides and polycyclic aromatic hydrocarbon (PAH) degradation.

Consortia of cyanobacteria/microalgae and bacteria in desert soils: an underexplored microbiota.

Desert ecosystem is generally considered as a lifeless habitat with extreme environmental conditions although it is colonized by extremophilic microorganisms. Cyanobacteria, microalgae, and bacteria in these habitats could tolerate harsh and rapidly fluctuating environmental conditions, intense ultraviolet radiation, and lack of water, leading to cell desiccation. They possess valuable metabolites withstanding extreme environmental conditions and make them good candidates for industrial applications. Moreover, most natural microorganisms in these extreme habitats exist as consortia that provide robustness and extensive metabolic capabilities enabling them to establish important relationships in desert environments. Engineering of such consortia of cyanobacteria, microalgae, and bacteria would be functional in the sustainable development of deserts through improving soil fertility, water preservation, primary production, pollutant removal, and maintaining soil stability. Modern tools and techniques would help in constructing highly functional cyanobacterial/microalgal-bacterial consortia that are greatly useful in the establishment of vegetation in deserts as well as in biotechnological applications.

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.

Use of mixed wastewaters from piggery and winery for nutrient removal and lipid production by Chlorella sp. MM3.

The larger-scale generation of piggery and winery wastewaters and consequent eutrophication are quite alarming, necessitating the use of a cost-effective treatment. This study attempted to remediate wastewaters from piggery and winery mixed in the ratios of 20:80, 50:50, 80:20, 100:0 and 0:100, in terms of nutrient removal and subsequent lipid accumulation by soil microalga, Chlorella sp. MM3. The per cent removal of total nitrogen and phosphates by the alga from mixed wastewaters within 10-days ranged between 51 and 89 and 26-49, respectively. As determined by FTIR spectroscopy, the lipid accumulation in the microalgal cells grown in wastewater mixtures ranged between 29 and 51%. Our results suggest that Chlorella sp. MM3 could be a potential candidate for bioremediation of wastewaters derived from piggery farm and winery industry, and that mixing of these wastewaters in 20:80 ratio would be an efficient approach for phycoremediation of mineral-rich effluents and subsequent yield of fairly good amounts of biofuel.

Comparison of plants with C3 and C4 carbon fixation pathways for remediation of polycyclic aromatic hydrocarbon contaminated soils.

The phytoremediation technique has been demonstrated to be a viable option for the remediation of polycyclic aromatic hydrocarbons (PAHs) contaminated sites. This study evaluated the potential applicability of plants with C3 and C4 carbon fixation pathways for the phytoremediation of recalcitrant high molecular weight (HMW) PAHs contaminated soil. A 60 and 120-day greenhouse study was conducted which showed higher degradation of HMW PAHs in soil grown with C4 plants when compared to C3 plants. Also, no PAHs were detected in the maize cobs, sunflower, wallaby, and Sudan grass seeds at the end of the experiment. The effect of plants in modifying the microbial community and dynamics in the rhizosphere was also examined by measuring soil biochemical properties such as dehydrogenase activity and water-soluble phenols. The results demonstrate a substantial difference in the microbial populations between planted and unplanted soils, which in turn facilitate the degradation of PAHs. To the best of our knowledge, this study for the first time evaluated the phytoremediation efficacy through the A. cepa cyto- and genotoxicity assay which should be considered as an integral part of all remediation experiments.