Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.     

Detection of Salmonella spp. and Listeria monocytogenes in suspended organic waste by nucleic acid extraction and PCR

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.     

Bacterial Population Dynamics in Dairy Waste during Aerobic and Anaerobic Treatment and Subsequent Storage

The objective of this study was to model a typical dairy waste stream, monitor the chemical and bacterial population dynamics that occur during aerobic or anaerobic treatment and subsequent storage in a simulated lagoon, and compare them to those of waste held without treatment in a simulated lagoon. Both aerobic and anaerobic treatment methods followed by storage effectively reduced the levels of total solids (59 to 68%), biological oxygen demand (85 to 90%), and sulfate (56 to 65%), as well as aerobic (83 to 95%), anaerobic (80 to 90%), and coliform (>99%) bacteria. However, only aerobic treatment reduced the levels of ammonia, and anaerobic treatment was more effective at reducing total sulfur and sulfate. The bacterial population structure of waste before and after treatment was monitored using 16S rRNA gene sequence libraries. Both treatments had unique effects on the bacterial population structure of waste. Aerobic treatment resulted in the greatest change in the type of bacteria present, with the levels of eight out of nine phyla being significantly altered. The most notable differences were the >16-fold increase in the phylum Proteobacteria and the approximately 8-fold decrease in the phylum Firmicutes. Anaerobic treatment resulted in fewer alterations, but significant decreases in the phyla Actinobacteria and Bacteroidetes, and increases in the phyla Planctomycetes, Spirochetes, and TM7 were observed.     

Paper chromatography and bioautography of L-carnitine and its acyl esters

A sensitive and specific bioautographic method for detecting l-carnitine and its derivatives has been doveloped, utilizing a mutant of Torulopsis bovina. As little as 10 ng of l-carnitine is detectable. Examples of applications of the method for detection of acylcarnitines in biological materials are presented.     

Odours and volatile organic compounds emitted from municipal solid waste at different stage of decomposition and relationship with biological stability

Odours (OU_E) and volatile organic compounds (VOC) emission during biological process used to treat MSW were studied under standardized conditions in order to detect potential risk for workers and population. Results obtained indicated that odours and VOCs emitted depend on the biological stability of waste measured by the dynamic respiration index (DRI) and a very good correlation were found between these parameters (OU_E vs. DRI, r = 0.96, p < 0.001, n = 6; VOC vs. DRI, r = 0.97, p < 0.001, n = 6).GC-MS study of the VOCs indicated the presence of a group of molecules that were degraded during the process. On the other hand, a second group of molecules, i.e. aromatic and halogenated compounds, and furan persisted in the waste sample, although molecule concentrations were always lower than Threshold Limit Value-Time Weighted Average (TLV-TWA).     

Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml⁻¹ and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml⁻¹. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.     

Silver Nanoparticles for the Adsorption of Manganese from Biological Samples

In this study, silver nanoparticles were prepared and used for separation and preconcentration of manganese from biological samples. The technical feasibility of silver nanoparticles for manganese removal was investigated under batch studies. The effects of different parameters such as pH of solution, time (t), amounts of PAN (E), and silver nanoparticles (N) on the adsorption of manganese by silver nanoparticle were investigated using factorial design and response surface methodology based on Box–Behnken design. Thermodynamic parameters indicate the adsorption process to be exothermic. The limit of detection of the proposed method followed by inductively coupled plasma was found to be 0.08?µg L^?1. The method was applied to determine of manganese in biological samples.     

A comparative life cycle assessment of two treatment technologies for the Grey Lanaset G textile dye: biodegradation by Trametes versicolor and granular activated carbon adsorption

Purpose

The aim of this study is to use life cycle assessment (LCA) to compare the relative environmental performance of the treatment using Trametes versicolor with a common method such as activated carbon adsorption. This comparison will evaluate potential environmental impacts of the two processes. This work compiles life cycle inventory data for a biological process that may be useful for other emergent biotechnological processes in water and waste management. LCA was performed to evaluate the use of a new technology for the removal of a model metal-complex dye, Grey Lanaset G, from textile wastewater by means of the fungus T. versicolor. This biological treatment was compared with a conventional coal-based activated carbon adsorption treatment to determine which alternative is preferable from an environmental point of view.

Materials and methods

The study is based on experimental research that has tested the novel process at the pilot scale. The analysis of the biological system ranges from the production of the electricity and ingredients required for the growth of the fungus and ends with the composting of the residual biomass from the process. The analysis of the activated carbon system includes the production of the adsorbent material and the electricity needed for the treatment and regeneration of the spent activated carbon. Seven indicators that measure the environmental performance of these technologies are included in the LCA. The indicators used are climate change, ozone depletion, human toxicity, photochemical oxidant formation, terrestial acidification, freshwater eutrophication, marine eutrophication, terrestrial ecotoxicity, freshwater ecotoxicity, marine ecotoxicity, metal depletion and fossil depletion.

Results

The results show that the energy use throughout the biological process, mainly for sterilisation and aeration, accounts for the major environmental impacts with the inoculum sterilisation being the most critical determinant. Nevertheless, the biological treatment has lower impacts than the physicochemical system in six of these indicators when steam is generated directly on site. A low-grade carbon source as an alternative to glucose might contribute to reduce the eutrophication impact of this process.

Conclusions

The LCA shows that the biological treatment process using the fungus T. versicolor to remove Grey Lanaset G offers important environmental advantages in comparison with the traditional activated carbon adsorption method. This study also provides environmental data and an indication of the potential impacts of characteristic processes that may be of interest for other applications in the field of biological waste treatment and wastewater treatment involving white-rot fungi.     

Use of Vero cell line to verify the biodetoxification efficiency of castor bean waste

Ricin is a toxic protein present in castor bean seeds (Ricinus communis). A toxic residue named castor bean waste is generated during biodiesel production process, such as that developed by PETROBRAS (the national petroleum company of Brazil). Solid-state fermentation (SSF) was used to detoxify castor bean waste through the Penicillium simplicissimum growth. After 24 h of fungal growth, the ricin was no longer identified by Sephadex G-50 gel chromatography. In order to verify the biological activity of ricin after several treatment stages, an in vitro assay using Vero cell line was carried out. Through this methodology, it was verified that after 24 and 48 h of treatment, the cell culture showed slightly growth inhibition. The waste was completely detoxified only after 72 h of fungal growth. This fact shows that an in vitro assay is important to verify the real efficiency of detoxification. Moreover, a relationship between the fungal protease production and the waste detoxification was observed.     

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2_IIA3_IIB_IIA1_II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

Volatile compounds flavoring obtained from Brazilian and Mexican spirit wastes by yeasts

Vinasse is a waste obtained from the production of beverages, such as tequila and cachaça. The presence of acids, alcohols, sugars, minerals, amino acids, peptides, and nitrogen salts make vinasse a hazardous liquid waste to the environment, affecting the fauna, flora, and microbiota of rivers and lagoons. This study used biological treatment concomitant to volatile compound production. The yeasts used in the study were Saccharomyces cerevisiae (CCMA 0187 and CCMA 0188), Candida parapsilosis (CCMA 0544), and Pichia anomala (CCMA 0193). A higher percentage reduction in chemical and biochemical oxygen demand was observed in the tequila vinasse than in the cachaça vinasse. However, a higher production of volatile compounds was observed in the cachaça vinasse. C. parapsilosis CCMA 0544 produced the highest concentration of 2-phenylethanol (162 mg L^?1). These results indicated that the environmental damage of vinasse can be reduced by treating vinasse with yeasts, and this treatment produces aroma compounds. This biological treatment has high economic potential, especially for the tequila industry.     

Lichens as suitable indicators of the biological effects of atmospheric pollutants around a municipal solid waste incinerator (S Italy)

A comprehensive biomonitoring programme should integrate several methods distributed along the biomonitoring chain, allowing to detect exposure, threads and impacts. In the case of a municipal solid waste incinerator (MSWI), biomonitoring of air pollution can contribute to source attribution, detection of ongoing processes and assessment of environmental effects. Three different methods were used to assess the biological effects of air pollution around a MSWI using lichens as biomonitors: (1) lichen diversity; (2) bioaccumulation of trace elements; and (3) physiological status (photosynthetic efficiency, cell membrane damage, viability). The first method takes into account the native lichen flora, while the other two were applied to thalli of the lichen Evernia prunastri transplanted for 6 months in the study area. Lichen diversity and physiological parameters reflected the effects of air pollution around the incinerator and the surrounding industrial area. High frequencies of non-nitrophilous species corresponded to sites with higher environmental quality, while high frequencies of nitrophilous species corresponded to sites with higher level of eutrophication. Transplanted samples showed increased cell membrane damage and reduced vitality respect to control samples. Bioaccumulation of trace elements pointed at the atmospheric origin of Hg depositions in the area. These results suggest that an integrated use of lichen-based methods along the biomonitoring chain can provide useful biological outputs for decision-makers to establish correct sustainable waste management policies.     

Precursor ion discovery on a hybrid quadrupole-time-of-flight mass spectrometer for gonadotropin-releasing hormone detection in complex biological mixtures

The gonadotropin-releasing hormone (GnRH) family includes several hypophysiotropic peptides occupying a central position in the regulatory loop controlling reproduction. Studies are still under way to clarify its biological role and evolutionary implication. Although sequencing of multiple genomes is bringing further advances in the understanding of the evolution of GnRH, there is still a need for biochemical studies aiming to identify GnRH from different species. Using a hybrid quadrupole-time-of-flight (Q-TOF) instrument, a new method for selective and sensitive GnRH detection and characterization from tissue extracts has been developed. The method uses the “precursor ion discovery” mode based on the capability of the Q-TOF analyzer to quickly record alternate mass spectra at low and high collision energy of precursor and product ion spectra, respectively, following liquid chromatographic separation of complex biological mixtures. The method exploits the selective detection of a specific b₂ product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine, highly conserved among nearly all species (22 of 24), and deriving from the preferential fragmentation of GnRHs carrying the dipeptide. Importantly, the method also includes acquisition of the product ion spectra from any candidate precursor ion, thereby allowing the determination of sequence information to confirm the GnRH identity or to isolate new ones.     

A new and versatile method for determination of thiolamines of biological importance

A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C₁₈ reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, N^ϵ-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.     

Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from Thermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaq Gold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, and Tfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima (Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTth from Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.     

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10⁻⁶ g of wet pig feces in 500 ml of phosphate-buffered saline and 10⁻⁴ g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

Cocultivation of Lactococcus lactis and Teredinobacter turnirae for biological chitin extraction from prawn waste

In this study, mainly biological treatment of prawn waste for chitin production was investigated. Lactic acid and protease fermentations were applied to extract chitin from prawn waste in the presence of various glucose concentrations. The results obtained were also compared with those of chemical method which was consisted of first mineral removal and then protein removal sequence. Different strategies were applied using lactic acid producing bacterium, Lactococcus lactis, and a protease producer, marine bacterium Teredinobacter turnirae. Both bacteria were first cultivated individually and then cofermented. In their individual cultivation, L. lactis removed the inorganic materials efficiently, while T. turnirae performed better in deproteinization process. Cofermentation of both bacteria was also conducted using three different protocols. The highest process yield (95.5%) was obtained when T. turnirae was first inoculated. Although the extraction of chitin by biological treatment was incomplete compared to the chemical method, the biological treatment employed here could still be considered as an alternative method in a more environmentally benign approach.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n = 65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10³ CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.