Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants.

Traditional methods for the enrichment of microorganisms rely on growth in a selective liquid medium or on an agar plate, followed by tedious characterization. Droplet microfluidic techniques have been recently used to cultivate microorganisms and preserve enriched bacterial taxonomic diversity. However, new methods are needed to select droplets comprising not only growing microorganisms but also those exhibiting specific properties, such as the production of value-added compounds. We describe here a droplet microfluidic screening technique for the functional selection of biosurfactant-producing microorganisms, which are of great interest in the bioremediation and biotechnology industries. Single bacterial cells are first encapsulated into picoliter droplets for clonal cultivation and then passively sorted at high throughput based on changes in interfacial tension in individual droplets. Our method expands droplet-based microbial enrichment with a novel approach that reduces the time and resources needed for the selection of surfactant-producing bacteria.

Bioprospecting CAZymes repertoire of Aspergillus fumigatus for eco-friendly value-added transformations of agro-forest biomass.

BACKGROUND: Valorizing waste residues is crucial to reaching sustainable development goals and shifting from a linear fossil-based economy to a circular economy. Fungal cell factories, due to their versatility and robustness, are instrumental in driving the bio-transformation of waste residues. The present work isolated a potent strain, i.e., Aspergillus fumigatus (ZS_AF), from an ancient Zloty Stok gold mine, which showcased distinctive capabilities for efficient hydrolytic enzyme production from lignocellulosic wastes. RESULTS: The present study optimized hydrolytic enzyme production (cellulases, xylanases, and ß-glucosidases) from pine sawdust (PSD) via solid-state fermentation using Aspergillus fumigatus (ZS_AF). The optimization, using response surface methodology (RSM), produced a twofold increase with maximal yields of 119.41 IU/gds for CMCase, 1232.23 IU/gds for xylanase, 63.19 IU/gds for ß-glucosidase, and 31.08 IU/gds for FPase. The secretome profiling validated the pivotal role of carbohydrate-active enzymes (CAZymes) and auxiliary enzymes in biomass valorization. A total of 77% of carbohydrate-active enzymes (CAZymes) were constituted by glycoside hydrolases (66%), carbohydrate esterases (9%), auxiliary activities (3%), and polysaccharide lyases (3%). The saccharification of pretreated wheat straw and PSD generated high reducing sugar yields of 675.36 mg/g and 410.15 mg/g, respectively. CONCLUSION: These findings highlight the significance of an efficient, synergistic, and cost-effective arsenal of fungal enzymes for lignocellulosic waste valorization and their potential to contribute to waste-to-wealth creation through solid-waste management. The utilization of Aspergillus fumigatus (ZS_AF) from an unconventional origin and optimization strategies embodies an innovative approach that holds the potential to propel current waste valorization methods forward, directing the paradigm toward improved efficiency and sustainability.

Recycling of both resin and fibre from wind turbine blade waste via small molecule-assisted dissolution.

Wind energy has significant growth potential and applicability on a global scale, but approximately 2.4% of wind turbine blades must be decommissioned annually. The majority of blade components can be recycled; however, wind blades are rarely recycled. In the present study, an alternative method was presented involving a small molecule-assisted technique based on a dynamic reaction that dissolves waste composite materials containing ester groups to recycle end-of-life wind turbine blades. This effective process requires temperatures below 200 °C, and the major component, i.e., resin, can be easily dissolved. This method can be applied to recycle composite materials, such as wind turbine blades and carbon fibre composites comprising fibres and resins. Depending on the waste, up to 100% of the resin degradation yield can be achieved. The solution used for the recycling process may be reused multiple times and can be reused to obtain resin-based components and create a closed loop for this type of material.

Decreasing lactate input for cost-effective sulfidogenic metal removal in sulfate-rich effluents: Mechanistic insights from (bio)chemical kinetics to microbiome response.

High material cost is the biggest barrier for the industrial use of low-molecular-weight organics (i.e. lactate) as external carbon and electron source for sulfidogenic metal removal in sulfate-rich effluents. This study aims to provide mechanistic evidence from kinetics to microbiome analysis by batch modeling to support the possibility of decreasing the lactate input to achieve cost-effective application. The results showed that gradient COD/SO42- ratios at a low level had promising treatment performance, reaching neutralized pH with nearly total elimination of COD (91%-99%), SO42- (85%-99%), metals (80%-99%) including Cu, Zn, and Mn. First-order kinetics exhibited the best fit (R2 = 0.81-0.98) to (bio)chemical reactions, and the simulation results revealed that higher COD/SO42- accelerated the reaction rate of SO42- and COD but not suitable to that of metals. On the other hand, we found that the decreasing COD/SO42- ratio increased average path distance but decreased clustering coefficient and heterogeneity in microbial interaction network. Genetic prediction found that the sulfate-reduction-related functions were significantly correlated with the reaction kinetics changed with COD/SO42- ratios. Our study, combining reaction kinetics with microbiome analysis, demonstrates that the use of lactate as a carbon source under low COD/SO42- ratios entails significant efficiency of metal removal in sulfate-rich effluent using SRB-based technology. However, further studies should be carried out, including parameter-driven optimization and life cycle assessments are necessary, for its practical application.

Bioprospecting of the Antarctic Bacillus subtilis strain for potential application in leaching hydrocarbons and trace elements from contaminated environments based on functional and genomic analysis.

The production of secondary metabolites including biosurfactants by the Bacillus subtilis ANT_WA51 and the evaluation of its ability to leach metals and petroleum derivatives from the soil, using post-culture medium was investigated. The ANT_WA51 strain isolated from a pristine, harsh Antarctic environment produces the biosurfactants surfactin and fengycin, which reduce the surface tension of molasses-based post-culture medium to 26.6 mN m-1 at a critical micellization concentration (CMC) of 50 mg L-1 and a critical micelle dilution (CMD) of 1:19. The presence of biosurfactants and other secondary metabolites in the post-culture medium contributed to significant removal of xenobiotics from contaminated soils in the batch washing experiment - 70% hydrocarbons and 10-23% metals (Zn, Ni and Cu). The isolate's tolerance to different abiotic stresses, including freezing, freeze-thaw cycles, salinity (up to 10%), the presence of metals - Cr(VI), Pb(II), Mn(II), As(V) (up to 10 mM) and Mo(VI) (above 500 mM) and petroleum hydrocarbons (up to 20.000 mg kg-1) as well as the confirmed metabolic activity of these bacteria in toxic environments in the OxiTop® system indicate that they can be used directly in bioremediation. Comparative genomic analysis of this bacteria revealed a high similarity of its genome to the associated plant strains from America and Europe indicating the wide applicability of plant growth-promoting Bacillus subtilis and that the data can be extrapolated to a wide range of environmental strains. An important aspect of the study was to present the absence of inherent features which would indicate its clear pathogenicity enables its safe use in the environment. Based on the obtained results, we also conclude that the use of post-culture medium, obtained on low-cost byproducts like molasses, for leaching contaminants, especially hydrocarbons, is a promising bioremediation method that can be a replacement for the use of synthetic surfactants and provides a base for further large-scale research but the selection of an appropriate leaching may be dependent on the concentration of contaminants.

Use of lignocellulosic waste materials in the passive treatment of highly alkaline wastewater contaminated with sulfates and metals - From a laboratory study to pilot scale.

Passive wastewater treatment systems are an alternative to costly and ineffective chemical wastewater treatment methods. Lignocellulosic waste materials (LWM) are often used in passive wastewater treatment systems as a cheap and accessible source of nutrients. LWM, such as spent mushroom compost and woodchips, have been implemented for the successful management of mildly alkaline effluents, which constitute a large fraction of industrial wastewater. The objective of the study was to provide an extensive study of the parameters in four types of commonly used LWM (raw and composted sawdust, spent mushroom compost and woodchips), which can be used in the planning of a passive wastewater treatment plant. LWM were shown to remove up to 90% Zn2+ and Pb2+ from a model solution and neutralize wastewater. Moreover, the LWM were inhabited by a physiologically diverse microbial consortium containing sulfate-reducing and cellulolytic microbes, which can influence the treatment process. Another purpose of this study was to construct a pilot wastewater treatment plant based on the use of LWM and gravel and to present its ability to effectively treat extremely alkaline flotation wastewater (pH = 12) originating from a lead and zinc mine located in Montenegro. The treated wastewater had a unique, but challenging chemical composition for passive treatment, as it was heavily contaminated with sulfates (∼1200 mg/L) and lead (∼1 g/L). The removal within the developed installation reached a rate of 66%, while the treated effluent, after initial neutralization, was maintained at a pH of approximately 7. Lead and zinc concentrations after treatment were also kept at levels required by Montenegrin law for wastewater disposal.

Mechanisms of NO2 Detection in Hybrid Structures Containing Reduced Graphene Oxide: A Review.

The sensitive detection of harmful gases, in particular nitrogen dioxide, is very important for our health and environment protection. Therefore, many papers on sensor materials used for NO2 detection have been published in recent years. Materials based on graphene and reduced graphene oxide deserve special attention, as they exhibit excellent sensor properties compared to the other materials. In this paper, we present the most recent advances in rGO hybrid materials developed for NO2 detection. We discuss their properties and, in particular, the mechanism of their interaction with NO2. We also present current problems occuring in this field.

Colorful Treasure From Agro-Industrial Wastes: A Sustainable Chassis for Microbial Pigment Production.

Colors with their attractive appeal have been an integral part of human lives and the easy cascade of chemical catalysis enables fast, bulk production of these synthetic colorants with low costs. However, the resulting hazardous impacts on the environment and human health has stimulated an interest in natural pigments as a safe and ecologically clean alternative. Amidst sources of natural producers, the microbes with their diversity, ease of all-season production and peculiar bioactivities are attractive entities for industrial production of these marketable natural colorants. Further, in line with circular bioeconomy and environmentally clean technologies, the use of agro-industrial wastes as feedstocks for carrying out the microbial transformations paves way for sustainable and cost-effective production of these valuable secondary metabolites with simultaneous waste management. The present review aims to comprehensively cover the current green workflow of microbial colorant production by encompassing the potency of waste feedstocks and fermentation technologies. The commercially important pigments viz. astaxanthin, prodigiosin, canthaxanthin, lycopene, and ß-carotene produced by native and engineered bacterial, fungal, or yeast strains have been elaborately discussed with their versatile applications in food, pharmaceuticals, textiles, cosmetics, etc. The limitations and their economic viability to meet the future market demands have been envisaged. The most recent advances in various molecular approaches to develop engineered microbiological systems for enhanced pigment production have been included to provide new perspectives to this burgeoning field of research.

Life cycle assessment and cost analysis for copper hydrometallurgy industry in China.

Understanding the environmental and economic impacts of copper hydrometallurgy throughout the whole life cycle is necessary for sustainable development of the copper industry. In this study, the environmental impacts and economic costs throughout the two major copper hydrometallurgical routes in China, including heap leaching and heap-agitation leaching, are analyzed and compared using the life cycle assessment (LCA) and life cycle cost (LCC) technique. The life cycle inventory compiled from the annual statistics of the Muliashi Copper Mine, and the data regarding energy and materials process are based on the GaBi databases. The environmental impacts are quantified into 12 indicators. The results show that compared with heap leaching route, heap-agitation leaching route reduces 36.8% of abiotic depletion potential (ADP elements), but increases over half of cumulative energy demand (CED), marine aquatic ecotoxicity potential (MAETP) and human toxicity potential (HTP). Furthermore, the stage of electrowinning and agitation leaching contributes the largest environmental impact to heap leaching and heap-agitation leaching route, respectively. This is mainly due to huge consumption of electricity and sulfuric acid. The analysis of economic cost reveals that heap leaching route needs internal cost of $3225/t Cu and external cost of $426/t Cu. Compared with heap leaching route, heap-agitation leaching route increased the internal and external cost by 18.9% and 54.2%, respectively. But the economic return from heap-agitation leaching is double that from heap leaching. Together, these results indicate heap-agitation leaching has a larger environmental impact and higher economic benefit than heap leaching, which is helpful for the government to design ecological compensation policies in the balance between ecological environment and economic development.

Insight Into Ecology, Metabolic Potential, and the Taxonomic Composition of Bacterial Communities in the Periodic Water Pond on King George Island (Antarctica).

Polar regions contain a wide variety of lentic ecosystems. These include periodic ponds that have a significant impact on carbon and nitrogen cycling in polar environments. This study was conducted to assess the taxonomic and metabolic diversity of bacteria found in Antarctic pond affected by penguins and sea elephants and to define their role in ongoing processes. Metabolic assays showed that of the 168 tested heterotrophic bacteria present in the Antarctic periodic pond, 96% are able to degrade lipids, 30% cellulose, 26% proteins, and 26% starch. The taxonomic classification of the obtained isolates differs from that based on the composition of the 16S rRNA relative abundances in the studied pond. The dominant Actinobacteria constituting 45% of isolates represents a low proportion of the community, around 4%. With the addition of run-off, the proportions of inhabiting bacteria changed, including a significant decrease in the abundance of Cyanobacteria, from 2.38 to 0.33%, increase of Firmicutes from 9.32 to 19.18%, and a decreasing richness (Chao1 index from 1299 to 889) and diversity (Shannon index from 4.73 to 4.20). Comparative studies of communities found in different Antarctic environments indicate a great role for penguins in shaping bacterial populations.

Comparative Genomics Provides Insights into the Genetic Diversity and Evolution of the DPANN Superphylum.

DPANN is known as highly diverse, globally widespread, and mostly ectosymbiotic archaeal superphylum. However, this group of archaea was overlooked for a long time, and there were limited in-depth studies reported. In this investigation, 41 metagenome-assembled genomes (MAGs) belonging to the DPANN superphylum were recovered (18 MAGs had average nucleotide identity [ANI] values of <95% and a percentage of conserved proteins [POCP] of >50%, while 14 MAGs showed a POCP of <50%), which were analyzed comparatively with 515 other published DPANN genomes. Mismatches to known 16S rRNA gene primers were identified among 16S rRNA genes of DPANN archaea. Numbers of gene families lost (mostly related to energy and amino acid metabolism) were over three times greater than those gained in the evolution of DPANN archaea. Lateral gene transfer (LGT; ∼45.5% was cross-domain) had facilitated niche adaption of the DPANN archaea, ensuring a delicate equilibrium of streamlined genomes with efficient niche-adaptive strategies. For instance, LGT-derived cytochrome bd ubiquinol oxidase and arginine deiminase in the genomes of "Candidatus Micrarchaeota" could help them better adapt to aerobic acidic mine drainage habitats. In addition, most DPANN archaea acquired enzymes for biosynthesis of extracellular polymeric substances (EPS) and transketolase/transaldolase for the pentose phosphate pathway from Bacteria. IMPORTANCE The domain Archaea is a key research model for gaining insights into the origin and evolution of life, as well as the relevant biogeochemical processes. The discovery of nanosized DPANN archaea has overthrown many aspects of microbiology. However, the DPANN superphylum still contains a vast genetic novelty and diversity that need to be explored. Comprehensively comparative genomic analysis on the DPANN superphylum was performed in this study, with an attempt to illuminate its metabolic potential, ecological distribution and evolutionary history. Many interphylum differences within the DPANN superphylum were found. For example, Altiarchaeota had the biggest genome among DPANN phyla, possessing many pathways missing in other phyla, such as formaldehyde assimilation and the Wood-Ljungdahl pathway. In addition, LGT acted as an important force to provide DPANN archaeal genetic flexibility that permitted the occupation of diverse niches. This study has advanced our understanding of the diversity and genome evolution of archaea.

Biostimulation of sulfate-reducing bacteria used for treatment of hydrometallurgical waste by secondary metabolites of urea decomposition by Ochrobactrum sp. POC9: From genome to microbiome analysis.

Sulfate-reducing bacteria (SRB) are key players in many passive and active systems dedicated to the treatment of hydrometallurgical leachates. One of the main factors reducing the efficiency and activity of SRB is the low pH and poor nutrients in leachates. We propose an innovative solution utilizing biogenic ammonia (B-NH3), produced by urea degrading bacteria, as a pretreatment agent for increasing the pH of the leachate and spontaneously stimulating SRB activity via bacterial secondary metabolites. The selected strain, Ochrobactrum sp. POC9, generated 984.7 mg/L of ammonia in 24 h and promotes an effective neutralization of B-NH3. The inferred metabolic traits indicated that the Ochrobactrum sp. POC9 can synthesize a group of vitamins B, and the production of various organic metabolites was confirmed by GC-MS analysis. These metabolites comprise alcohols, organic acids, and unsaturated hydrocarbons that may stimulate biological sulfate reduction. With the pretreatment of B-NH3, sulfate removal efficiency reached ~92.3% after 14 days of incubation, whereas SRB cell count and abundance were boosted (~107 cell counts and 88 OTUs of SRB) compared to synthetic ammonia (S-NH3) (~103 cell counts and 40 OTUs of SRB). The dominant SRB is Desulfovibrio in both S-NH3 and B-NH3 pretreated leachate, however, it belonged to two different clades. By reconstructing the ecological network, we found that B-NH3 not only directly increases SRB performance but also promotes other strains with positive correlations with SRB.

Microbiological Sulfide Removal-From Microorganism Isolation to Treatment of Industrial Effluent.

Management of excessive aqueous sulfide is one of the most significant challenges of treating effluent after biological sulfate reduction for metal recovery from hydrometallurgical leachate. The main objective of this study was to characterize and verify the effectiveness of a sulfide-oxidizing bacterial (SOB) consortium isolated from post-mining wastes for sulfide removal from industrial leachate through elemental sulfur production. The isolated SOB has a complete sulfur-oxidizing metabolic system encoded by sox genes and is dominated by the Arcobacter genus. XRD analysis confirmed the presence of elemental sulfur in the collected sediment during cultivation of the SOB in synthetic medium under controlled physicochemical conditions. The growth yield after three days of cultivation reached ~2.34 gprotein/molsulfid, while approximately 84% of sulfide was transformed into elemental sulfur after 5 days of incubation. Verification of isolated SOB on the industrial effluent confirmed that it can be used for effective sulfide concentration reduction (~100% reduced from the initial 75.3 mg/L), but for complete leachate treatment (acceptable for discharged limits), bioaugmentation with other bacteria is required to ensure adequate reduction of chemical oxygen demand (COD).

Mobile genetic elements mediate the mixotrophic evolution of novel Alicyclobacillus species for acid mine drainage adaptation.

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions.

Characterization of Graphite Oxide and Reduced Graphene Oxide Obtained from Different Graphite Precursors and Oxidized by Different Methods Using Raman Spectroscopy Statistical Analysis.

In this paper, various graphite oxide (GO) and reduced graphene oxide (rGO) preparation methods are analyzed. The obtained materials differed in their properties, including (among others) their oxygen contents. The chemical and structural properties of graphite, graphite oxides, and reduced graphene oxides were previously investigated using Raman spectroscopy (RS), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). In this paper, hierarchical clustering analysis (HCA) and analysis of variance (ANOVA) were used to trace the directions of changes of the selected parameters relative to a preparation method of such oxides. We showed that the oxidation methods affected the physicochemical properties of the final products. The aim of the research was the statistical analysis of the selected properties in order to use this information to design graphene oxide materials with properties relevant for specific applications (i.e., in gas sensors).

Characterization of Fe-based sediments received from chemical pre-treatment of hydrometallurgical waste leachate from the recycling of alkaline batteries.

The waste leachate from the hydrometallurgical recycling of spent batteries contains a significant amount of undesirable iron that needs to be precipitated before the recovery of target metals. The produced Fe-sediments are usually disposed of or stored at the treatment site as waste and are often poorly managed. This work estimates the environmental stability and application potential of Fe-sediments produced from highly acidic hydrometallurgical leachate during the recycling of spent alkaline batteries. After pH neutralization of the leachate by Na2CO3, a primary Fe-sediment (PFS), mainly composed of highly unstable metal (i.e., Fe, Zn, and Mn) sulfates, was obtained. The subsequent rinsing of this unstable PFS sediment led to the production of a secondary Fe-sediment (SFS), which was composed of an amorphous-phased ferric iron sulfate hydrate - Fe16O16(SO4)3(OH)10·10H2O. The results of single extraction using chemical reagents and biological dissolution by iron-transforming bacteria confirmed that despite most of the ions in PFS were dissolvable, the processed SFS was environmentally safe. The sorption efficiency of SFS towards Pb(II) and As(V) (up to ~ 99% and 94%, respectively, with an initial concentration of 100 mg/L) was found to be promising, suggesting the high potential for economical reuse of SFS.

An integrated insight into bioleaching performance of chalcopyrite mediated by microbial factors: Functional types and biodiversity.

Six artificial communities with different function or biodiversity were reconstructed by six typical bioleaching species for chalcopyrite leaching. Absence of sulfur oxidizers in communities significantly reduced copper extraction rates, and low diversity communities also exhibited slightly poor bioleaching performances. The variations of pH, redox potential, ferrous and copper ions indicated that the community with both sulfur oxidizers and high diversity showed fast adaptation to the environment and rapid dissolution of chalcopyrite. Integrated analysis of mineralogical and microbial parameters demonstrated that functional types of microorganisms made more contributions in mediating chalcopyrite dissolution than microbial diversity. Further correlation analysis between microbial types and chalcopyrite dissolution performances showed that sulfur oxidizers, especially Acidithiobacillus caldus, could greatly accelerate chalcopyrite dissolution by regulating solution physicochemical factors, such as redox potential and pH. This study provided a theoretical basis for improving bioleaching efficiency by balancing microbial functional types and biodiversity.

Effect of Clinoptilolite and Halloysite Addition on Biogas Production and Microbial Community Structure during Anaerobic Digestion.

The study presents a comparison of the influence of a clinoptilolite-rich rock-zeolite (commonly used for improving anaerobic digestion processes)-and a highly porous clay mineral, halloysite (mainly used for gas purification), on the biogas production process. Batch experiments showed that the addition of each mineral increased the efficiency of mesophilic anaerobic digestion of both sewage sludge and maize silage. However, halloysite generated 15% higher biogas production during maize silage transformation. Halloysite also contributed to a much higher reduction of chemical oxygen demand for both substrates (by ~8% for maize silage and ~14% for sewage sludge) and a higher reduction of volatile solids and total ammonia for maize silage (by ~8% and ~4%, respectively). Metagenomic analysis of the microbial community structure showed that the addition of both mineral sorbents influenced the presence of key members of archaea and bacteria occurring in a well-operated biogas reactor. The significant difference between zeolite and halloysite is that the latter promoted the immobilization of key methanogenic archaea Methanolinea (belong to Methanomicrobia class). Based on this result, we postulate that halloysite could be useful not only as a sorbent for (bio)gas treatment methodologies but also as an agent for improving biogas production.

In vivo creation of plasmid pCRT01 and its use for the construction of carotenoid-producing Paracoccus spp. strains that grow efficiently on industrial wastes.

BACKGROUND: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media. RESULTS: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose. CONCLUSIONS: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.