Recycling air conditioner-generated condensate water for microalgal biomass production and carbon dioxide sequestration.

Air conditioners alleviate the discomfort of human beings from heat waves that are consequences of climate change caused by anthropogenic activities. With each passing year, the effects of global warming worsen, increasing the growth of air conditioning industry. Air conditioning units produce substantial amounts of non-nutritive and (generally) neglected condensate water and greenhouse gases. Considering this, the study explored the potential of using air conditioner condensate water (ACW) to cultivate Chlorella sorokiniana, producing biomass, and sequestering carbon dioxide (CO2). The maximum biomass production was obtained in the BG11 medium (1.45 g L-1), followed by ACW-50 (1.3 g L-1). Similarly, the highest chlorophyll-a content was observed in the BG11 medium (11 µg mL-1), followed by ACW-50 (9.11 µg mL-1). The ACW-50 cultures proved to be better adapted to physiological stress (Fv/Fm > 0.5) and can be suitable for achieving maximum biomass with adequate lipid, protein, and carbohydrate production. Moreover, C. sorokiniana demonstrated higher lipid and carbohydrate yields in the ACW-50 medium, while biomass production and protein yields were comparable to the BG11 medium. The lipid, protein, and carbohydrate productivity were 23.43, 32.9, and 23.19 mg L-1 d-1, respectively for ACW-50. Estimation of carbon capture potential through this approach equals to 9.5% of the total emissions which is an added advantage The results indicated that ACW could be effectively utilized for microalgae cultivation, reducing the reliance on freshwater for large-scale microalgal biomass production and reduce the carbon footprints of the air conditioning industry.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

A review on application of next-generation sequencing methods for profiling of protozoan parasites in water: Current methodologies, challenges, and perspectives.

The advancement in metagenomic techniques has provided novel tools for profiling human parasites in environmental matrices, such as water and wastewater. However, application of metagenomic techniques for the profiling of protozoan parasites in environmental matrices is not commonly reported in the literature. The key factors leading to the less common use of metagenomics are the complexity and large eukaryotic genome, the prevalence of small parasite populations in environmental samples compared to bacteria, difficulties in extracting DNA from (oo)cysts, and limited reference databases for parasites. This calls for further research to develop optimized methods specifically looking at protozoan parasites in the environment. This study reviews the current workflow, methods and provide recommendations for the standardization of techniques. The article identifies and summarizes the key methods, advantages, and limitations associated with metagenomic analysis, like sample pre-processing, DNA extraction, sequencing approaches, and analysis methods. The study enhances the understanding and application of standardized protocols for profiling of protozoan parasite community from highly complexe samples and further creates a resourceful comparison among datasets without any biases.

Microplastics in the environment: Interactions with microbes and chemical contaminants.

Microplastics (MPs) are contaminants of emerging concern that have gained considerable attention during the last few decades due to their adverse impact on living organisms and the environment. Recent studies have shown their ubiquitous presence in the environment including the atmosphere, soil, and water. Though several reviews have focused on the occurrence of microplastics in different habitats, little attention has been paid to their interaction with biological and chemical pollutants in the environment. This review therefore presents the state of knowledge on the interaction of MPs with chemicals and microbes in different environments. The distribution of MPs, the association of toxic chemicals with MPs, microbial association with MPs and the microbial-induced fate of MPs in the environment are discussed. The biodegradation and bioaccumulation of MPs by and in microbes and its potential impact on the food chain are also reviewed. The mechanisms driving these interactions and how these, in turn, affect living organisms however are not yet fully understood and require further attention.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Concentration and reduction of antibiotic residues in selected wastewater treatment plants and receiving waterbodies in Durban, South Africa.

In the province of KwaZulu-Natal, South Africa the incidence of resistant tuberculosis, upper respiratory tract diseases as well as diarrhoeal and parasitic infections is high. Treatment of these diseases with antibiotics is partly reflected by the excretion of the respective antibiotics and their subsequent occurrence in wastewater. Their quantitative reduction in wastewater treatment reflects their potential environmental as well as human impact, the latter due to the use of the recipient water for domestic purposes and for irrigation. Information of the occurrence and reduction of different classes of antibiotics in wastewater treatment is sparse, especially the particle bound fraction of these. Due to this, analyses of aqueous and particle bound antibiotics in untreated wastewater of four selected wastewater treatment plants (WWTPs) and their receiving water bodies was carried out in Durban, South Africa. The treatment step especially considered was the biological one, represented by activated sludge and trickling filters. The treatment further included secondary clarifiers and final chlorine disinfection. Composite samples were collected during the period February 2017 to May 2017 and analysed with online solid phase extraction - high performance liquid chromatography mass spectrometry (SPE-HPLC-MS). For the 13 assessed antibiotics, the limit of detection (LOD) and the limit of quantification (LOQ) ranged from 0.07 to 0.33 ng L-1 and 0.23 to 1.09 ng L-1 respectively, while the total percentage recovery was in the range of 51 to 111%. The percentage of individual antibiotics bound to the particulate fraction normally lost by sample (influent) filtration, if not analysed in parallel, was in the range of 2.6%-97.3% (n = 32). In this fraction (sludge from centrifuge sample), the concentration of bound antibiotics of all the target antibiotics were detected in the influent of all WWTP in concentration ranges between 1.3 ng L-1 (Azithromycin; AZI) to 81,748 ng L-1 (Ciprofloxacin; CIP). The antibiotics with the highest median concentrations in receiving water bodies of the respective WWTP were; Sulfamethoxazole; SUL (239 ng L-1) WWTP "K", Ciprofloxacin; CIP (708 ng L-1) WWTP "S" and Albendazole; ALB (325 ng L-1 and 683 ng L-1) WWTP "P" and "I" respectively. The overall percentage removal efficiency for the four WWTPs ranged from 21% to 100%. The biological treatment steps, activated sludge and trickling filters, were effective in removing antibiotics especially with the trickling filter and the impact of the sedimentation stage after activated sludge treatment.

Identification of antibiotics in wastewater: current state of extraction protocol and future perspectives.

The release and occurrence of antibiotics in the aquatic environment has generated increased attention in the past few decades. The residual antibiotic in wastewater is important in the selection for antimicrobial resistance among microorganisms and the possibility of forming toxic derivatives. This review presents an assessment of the advancement in methods for extraction of antibiotics with solid phase extraction and liquid-liquid extraction methods applied in different aquatic environmental media. These advanced methods do enhance specificity, and also exhibit high accuracy and recovery. The aim of this review is to assess the pros and cons of the methods of extraction towards identification of quinolones and sulphonamides as examples of relevant antibiotics in wastewater. The challenges associated with the improvements are also examined with a view of providing potential perspectives for better extraction and identification protocols in the near future. From the context of this review, magnetic molecular imprinted polymer is superior over the remaining extraction methods (with the availability of commercial templates and monomers), is based on less cumbersome extraction procedures, uses less solvent and has the advantage of its reusable magnetic phase.

Evaluation of phytotoxicity effect on selected crops using treated and untreated wastewater from different configurative domestic wastewater plants.

This study investigated the phytotoxicity effect of untreated and treated wastewater collected from two different configurations of domestic wastewater treatment plants in South Africa. The phytotoxicity effect on vegetable seed growth was studied in terms of germination index (GI), relative seed germination (RSG) and relative root elongation (RRE) using four commercial crop varieties, viz., tomato (Lycopersicon esculentum), radish (Raphanus sativus), carrot (Daucus carota) and onion (Allium cepa). According to phyototoxicity limits, 80% germination and above is regarded as non-toxic and less than 50% GI is regarded as highly toxic and not suitable for agricultural purposes. In our study, seeds were irrigated with concentrations of 25%, 50%, 75%, 100% of treated effluent (TE) and untreated effluent (UTE). The TE results were best with the highest GI (%) recorded as tomato, 177; carrot, 158.5; onion, 132; and lettuce, 124. The results of this study indicate that TE showed no phytotoxicty effects and recorded above 80% GI. The UTE irrigated crops reached a GI of only 50% and above which is clear evidence of the beneficial effect of waste water treatment. The overall results confirmed that treated wastewater has a beneficial effect on agricultural crops and can be used as a liquid fertilizer.

Development of poly(aspartic acid-co-malic acid) composites for calcium carbonate and sulphate scale inhibition.

Polyaspartic acid (PSI) is suitable for the inhibition of inorganic scale deposition. To enhance its scale inhibition efficiency, PSI was modified by reacting aspartic acid with malic acid (MA) using thermal polycondensation polymerization. This reaction resulted in poly(aspartic acid-co-malic acid) (PSI-co-MA) dual polymer. The structural, chemical and thermal properties of the dual polymers were analysed by using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry and gel permeation chromatography. The effectiveness of six different molar ratios of PSI-co-MA dual polymer for calcium carbonate and calcium sulphate scale inhibition at laboratory scale batch experiments was evaluated with synthetic brine solution at selected doses of polymer at 65-70°C by the static scale test method. The performance of PSI-co-MA dual polymer for the inhibition of calcium carbonate and calcium sulphate precipitation was compared with that of a PSI single polymer. The PSI-co-MA exhibited excellent ability to control inorganic minerals, with approximately 85.36% calcium carbonate inhibition and 100% calcium sulphate inhibition at a level of 10 mg/L PSI-co-MA, respectively. Therefore, it may be reasonably concluded that PSI-co-MA is a highly effective scale inhibitor for cooling water treatment applications.

Development of anti-scale poly(aspartic acid-citric acid) dual polymer systems for water treatment.

The formation of calcium sulphate and calcium carbonate scale poses major problems in heat exchangers and water cooling systems, thereby affecting the performance of these types of equipment. In order to inhibit these scale formations, new types of biodegradable water soluble single polymer and dual poly(aspartic acid-citric acid) polymers were developed and tested. The effectiveness of single polymer and four different compositions of poly aspartic acid and citric acid dual polymer systems as scale inhibitors were evaluated. Details of the synthesis, thermal stability, scale inhibition and the morphological characterization of single and dual polymers are presented in this scientific paper. It was found that the calcium sulphate scale inhibition rate was in the range 76.06-91.45%, while the calcium carbonate scale inhibition rate observed was in the range 23.37-30.0% at 65-70 °C. The finding suggests that the water soluble dual polymers are very effective in sulphate scale inhibition in comparison of calcium carbonate scale inhibition.

Improving the feasibility of producing biofuels from microalgae using wastewater.

Biofuels have received much attention recently owing to energy consumption and environmental concerns. Despite many of the technologies being technically feasible, the processes are often too costly to be commercially viable. The major stumbling block to full-scale production of algal biofuels is the cost of upstream and downstream processes and environmental impacts such as water footprint and indirect greenhouse gas emissions from chemical nutrient production. The technoeconomics of biofuels production from microalgae is currently unfeasible due to the cost of inputs and productivities achieved. The use of a biorefinery approach sees the production costs reduced greatly due to utilization of waste streams for cultivation and the generation of several potential energy sources and value-added products while offering environmental protection. The use of wastewater as a production media, coupled with CO2 sequestration from flue gas greatly reduces the microalgal cultivation costs. Conversion of residual biomass and by-products, such as glycerol, for fuel production using an integrated approach potentially holds the key to near future commercial implementation of biofuels production.

BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae.

In order to develop feasible production processes for microalgal biodiesel, the isolation of high neutral lipid producing microalgae is crucial. Since the established Nile Red (NR) method for detection of intracellular lipids has been successful only for some microalgae, a more broadly applicable detection method would be desirable. Therefore, BODIPY 505/515, a lipophilic bright green fluorescent dye was tested for detection of intracellular lipids in Chlorella vulgaris, Dunaliella primolecta and Chaetoceros calcitrans. An optimum concentration of 0.067 µg ml(-1) was determined for lipid staining in the microalgae. Compared to NR, BODIPY 505/515 was more effective in staining microalgae and showed resistance to photobleaching, maintaining its fluorescence longer than 30 min.

PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids.

This study investigated the use of Pulse Amplitude Modulated (PAM) fluorometry to measure nutrient induced physiological stress and subsequent synthesis of cellular neutral lipids. A freshwater Chlorella sp. was subjected to complete nutrient stress (distilled H2O) and selective nutrient stress in modified BG-11 media (BG-11-N, BG-11-P and BG-11-Fe). Physiological stress was recorded using parameters, rETR, Fv/Fm, Ek, α and NPQ. Induced stress became evident when these parameters were significantly altered, suggesting the onset of neutral lipid synthesis. Complete nutrient stress induced the highest yield of cellular neutral lipids (∼49%) compared to absence of selected nutrients (∼30%). Physiological stress was recorded by a significant decrease in rETR (75%), Fv/Fm (36%), and Ek (60%) and an increase in NPQ (83%). Optimization of neutral lipids occurred by initially maximizing the biomass and subsequently subjecting the harvested biomass to complete nutrient stress.

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production.

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.

Application of quantitative RT-PCR to determine the distribution of Microthrix parvicella in full-scale activated sludge treatment systems.

Three wastewater treatment plants in South Africa were investigated to understand the phylogeny and distribution of Microthrix parvicella using real-time polymerase chain reaction (RT-PCR). The phylogenetic analysis of the 16S rRNA of M. parvicella revealed 98% to 100% homology of South African clones to M. parvicella reported in Genbank. The standard curves for RT-PCR showed R2 values greater than 0.99, accurate for quantification. The relative occurrence of M. parvicella 16S rRNA gene copies in the three wastewater treatment plants was in the range 0% to 3.97%. M. parvicella copies increased when the environmental temperature (< or =20 degrees C) and food/microorganism (F/M) ratio was low. The M. parvicella 16S rRNA copies could be positively correlated to the sludge volume index at low temperature. At higher temperature, there was a rapid reduction in M. parvicella population irrespective of other favorable factors, indicating the strong influence of temperature on filamentous proliferation. RT-PCR has potential applications in wastewater treatment plants to monitor sudden shift in the microbial population and assessing the plants efficacy.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater.

A Kubotatrade mark submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

Comparative evaluation of the microbial community in biological processes treating industrial and domestic wastewaters.

AIMS: Comparison of the microbial composition and process performance between laboratory scale processes treating domestic and vegetable oil wastewaters. METHODS AND RESULTS: Two laboratory scale modified Ludzack-Ettinger processes were operated under similar operating conditions. One process was fed domestic wastewater and the other an industrial wastewater, vegetable oil effluent. Nitrogen removal capacities of the processes were similar. The industrial process exhibited a lower COD removal capacity and oxygen utilization rate, although a greater mixed liquor volatile suspended solids concentration was observed in the industrial process. Fluorescent in situ hybridization (FISH) with probes EUBmix, ALF1b, BET42a, GAM42a and HGC69a revealed that 81% and 72% of total cells stained with 4', 6-diamidino-2-phenylindole (DAPI) within the domestic and industrial processes respectively bound to EUBmix. This indicated a slightly lower Eubacterial population within the industrial process. The alpha-proteobacteria was the dominant community in the industrial process (31% of EUBmix), while the beta-proteobacteria dominated the domestic process (33% of EUBmix). CONCLUSIONS: The findings served to establish a difference in the microbial population between the processes. Therefore, the class alpha-proteobacteria could play a primary role in the degradation of vegetable oil effluent. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in process design and retrofitting of biological processes treating vegetable oil effluent.

Using respirometric techniques and fluorescent in situ hybridization to evaluate the heterotrophic active biomass in activated sludge.

The separation and accurate quantification of active biomass components in activated sludge is of paramount importance in models, used for the management and design of waste water (WW) treatment plants. Accurate estimates of microbial population concentrations and the direct, in situ determination of kinetic parameters could improve the calibration and validation of existing models of biological nutrient removal activated sludge systems. The aim of this study was to obtain correlations between heterotrophic active biomass (Z(BH)) concentrations predicted by mathematical models and quantitative information obtained by Fluorescent in situ hybridizations (FISH). Respirometric batch test were applied to mixed liquors drawn from a well-defined parent anoxic/aerobic activated sludge system to quantify the Z(BH) concentrations. Similarly fluorescent labeled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidizers were used in combination with DAPI staining to validate the Z(BH) active biomass component in activate sludge respirometric batch tests. For the direct enumeration and simultaneous in situ analysis of the distribution of nitrifying bacteria, in situ hybridization with oligonucleotide probes were used. Probes (NSO 1225, NSR 1156, and NIT3) were used to target the nitrifiers and the universal probe (EUB MIX) was used to target all Eubacteria. Deducting the lithoautotrophic population from the total bacteria population revealed the Z(BH) population. A conversion factor of 8.49 x 10(-11) mg VSS/cell was applied to express the Z(BH) in terms of COD concentration. Z(BH) values obtained by molecular probing correlated closely with values obtained from the modified batch test. However, the trend of consistently poor correspondence of measured and theoretical concentrations were evident. Therefore, the focus of this study was to investigate alternative technology, such as FISH to validate or replace kinetic parameters which are invariably incorporated into models.

Microbiological studies of an anaerobic baffled reactor: microbial community characterisation and deactivation of health-related indicator bacteria.

This WRC funded project has studied the appropriateness of the ABR (anaerobic baffled reactor) for on-site primary sanitation in low-income communities. A 3,000 L pilot reactor was located at the Kingsburgh wastewater treatment plant south of Durban, South Africa. Feed to the reactor was raw domestic wastewater containing a significant proportion of particulate organic matter. The compartments of the ABR were routinely monitored for pH, COD, and gas production, among other physical-chemical determinants. The microbial population in each compartment was analysed by fluorescent in situ hybridisation, using general oligonucleotide probes for eubacteria and archeae and a suite of 10 genera or family specific probes. Scanning electron microscopy was conducted on the sludge fraction of each compartment. Mixed fractions from each compartment were also analysed for health-related indicator bacteria (total coliforms and E. coli). Results indicated that methanogenesis was not occurring to the expected extent in the latter compartments, and that this was probably due to a hydraulic load limitation. This contrasted with earlier studies on industrial effluent, for which the organic load was exclusively in soluble form. Inactivation of health-related indicator bacteria was less than 1 log, indicating the need for an additional post-treatment of the effluent to protect community health.

Anoxic phosphorus removal by denitrifying heterotrophic bacteria.

The unexplained occurrence of anoxic phosphorus (P) accumulation has largely hampered modeling of nitrification denitrification biological excess P removal (NDBEPR) systems. The aim of this study was, therefore, to isolate and identify denitrifying-P accumulating heterotrophic bacteria (DPBs) from a NDBEPR system in order to evaluate anoxic P accumulation and the specific mechanisms involved. Results of the study showed various heterotrophic bacteria to be capable of anoxic P accumulation utilising nitrate (NO3) as electron acceptor. While Pseudomonas spp. predominated, Serratia spp. and Vibrio spp. demonstrated the most efficient anoxic P accumulation with 7.10 and 7.29 mgPO4-P/L removal, respectively, at an initial NO3 concentration of 13.54 mgNO3-N/L and P concentration of 16.34 mgPO4-P/L. Weaker DPBs were also identified which were only capable of accumulating small amounts of P at low initial P and NO3 concentrations due to weak denitrification capacity. Anoxic P release was also observed due to the presence of acetate.