Impact of Bioaugmentation with a Consortium of Bacteria on the Remediation of Wastewater-Containing Hydrocarbons (5 pp)

Goals, Scope and Background It has been observed that hydrocarbon treated wastewaters still contain high COD and a number of intermediates. This suggests that the required catabolic gene pool for further degradation might be absent in the system or, that its titer value is not significant enough. By providing the desired catabolic potential, the overall efficiency of the treatment system can be improved. This study aims to demonstrate this concept by bioaugmentation of a lab-scale reactor treating refinery wastewater with a consortium having the capacity to complement the alkB genotype to the available microbial population. Methods Two reactors were set up using activated biomass collected from a refinery treatment plant and operated at a continuous mode for a period of 8 weeks. The feed to both reactors was kept constant. Crude oil was spiked regularly. One reactor was bioaugmented with a consortium previously described for crude oil spill remediation. The efficiency of the bioaugmented reactor was demonstrated by reduced COD. The changes in the microbial population over a period of time were analyzed by RAPD. Catabolic activity of the biomass in both reactors was monitored by PCR. The presence of the catabolic loci was confirmed by Southern Hybridization. Results and Discussion 52.2% removal of COD was observed in the bioaugmented reactor while only 15.1% reduction of COD was observed in the reactor without bioaugmentation. The change in microbial population can be seen from the 4th week, which also corresponds to improved catabolic activity. The presence of the bedA locus was seen in all samples, which indicates the presence of aromatic degraders, but the appearance of the alkB locus, from the 6th week onwards, which was observed only in the samples from the bioaugmented reactor. The results suggest that the gene pool of the bioaugmented reactor has catabolic loci that can degrade accumulated intermediates, thus improving the efficiency of the system. Conclusions In this study, improvement of efficiency of bioremediation was demonstrated by addition of catabolic loci that are responsible for degradation. Bioaugmentation was carried out in biomass that was collected from an ETP (effluent treatment plant) treating hydrocarbon containing wastewater to study the strategies for improvement of the treatment system. Biostimulation, only marginally improved the efficiency, when compared to bioaugmentation. The improved efficiency was demonstrated by COD removal. The presence of the alkB locus suggests the importance of a catabolic gene pool that acts on accumulated intermediates. It is well documented that straight chain aliphatics and intermediates of aromatic compounds after ring cleavage, accumulate in refinery wastewater systems, thereby hindering further degradation of the wastewater. Supplementation of a catabolic gene pool that treats the lower pathway compounds and alkanes will improve the overall efficiency. In this study, results suggest that the alkB locus can also be used to monitor the degradative mode of the activated biomass. Recommendations and Perspective . Pollution from petroleum and petroleum products around the globe are known to have grave consequences on the environment. Bioremediation, using activated sludge, is one option for the treatment of such wastes. Effluent treatment plants are usually unable to completely degrade the wastewater being treated in the biological unit (the aerator chambers). The efficiency of degradation can be improved by biostimulation and bioaugmentation. This study demonstrates the improved efficiency of a treatment system for wastewater containing hydrocarbons by bioaugmentation of a consortium that supports degradation. Further experiments on a pilot scale are recommended to assess the use of bioaugmentation on a large scale. The use of molecular tools, like DNA probes for alkB, to monitor the system also needs to be explored.     

Heavy metals in untreated/treated urban effluent and sludge from a biological wastewater treatment plant

Background, Aim and Scope

The presence of heavy metals in wastewater is one of the main causes of water and soil pollution. The aim of the present study was to investigate the removal of Cd, Cu, Pb, Hg, Mn, Cr and Zn in urban effluent by a biological wastewater treatment, as well as to quantify the levels of As, Be, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sn, Tl, V and Zn in dewatering sludge from the Biological Wastewater Treatment Plant to Ribeirão Preto (RP-BWTP), Brazil.

Materials and Methods

Concentrations of Cd, Cr, Cu, Mn and Pb in wastewater and those of Ni in sludge were determined by atomic absorption spectrophotometry with graphite furnace atomization. Mercury concentrations in wastewater were measured by hydride generation atomic spectrophotometry, and Zn levels were determined by atomic absorption spectrophotometry using acetylene flame. In sludge, the levels of As, Be, Cd, Cr, Cu, Fe, Hg, Mn, Pb, Sn, Tl, V and Zn were determined by inductively coupled plasma-mass spectrometry.

Results

The percentages of removal efficiency (RE) were the following: Hg 61.5%, Cd 60.0%, Zn 44.9%, Cu 44.2%, PB 39.7%, Cr 16,5% and Mn 10.4%. In turn, the mean concentrations (mg/kg) of metals in dewatering sludge followed this increasing order: Tl (<0.03), Hg (0.31), Be (0.43), As (1.14), Cd (1.34), V (59.2), Pb (132.1), Sn (166.1), Cr (195.0), Mn (208.1), Ni (239.4), Cu (391.7), Zn (864.4) and Fe (20537).

Discussion

The relationship between metal levels in untreated wastewater, as well as the removal efficiency are in agreement with previous data from various investigators, It is important to note that metal removal efficiency is not only affected by metal ion species and concentration, but also by other conditions such as operating parameters, physical, chemical, and biological factors.

Conclusions

Metal values recorded for treated wastewater and sludge were within the maximum permitted levels established by the Environmental Sanitation Company (CETESB), São Paulo, Brazil.

Recommendations

There is an urgent need for the authorities who are responsible for legislation on sludge uses in agriculture of establishing safety levels for As, Be, Hg, Sn, Tl and V.

Perspectives

According to the current metal levels, RP-BWTP sludge might be used for agriculture purposes. However, for an environmentally safe use of sewage sludge, further studies including systematic monitoring are recommended. Annual metal concentrations and predicted variations of those elements in the sludge should be monitored.

     

Endpoint ‘floating leaves’ of <Emphasis Type="Italic">Potamogeton natans</Emphasis>: A new method to evaluate the development of macrophytes in pond mesocosms

Goal, Scope and Background One of the advantages of long-term mesocosm experiments as compared to short-term standard toxicity tests in the laboratory is the potential for detecting secondary effects due to the interaction of species and recovery with biomass of macrophytes being an important endpoint. However, generating biomass data by harvesting is often laborious, time-consuming, costly and restricted to the end of the experiment. Moreover, valuable information may get lost, in particular in single application studies, since maximal primary effects and secondary effects or recovery occur per se at different times. Potamogeton natans was used as an example in order to test whether number and area of floating leaves can be reliably measured and be used as intermediate and final endpoints in mesocosm effect studies. Methods Digital photos, which were taken of the water surface in the course of an indoor pond mesocosm study on herbicide effects, were subjected to image analysis. The results were compared to wet weight and ash-free dry weight of Potamogeton at the end of the herbicide study. Results and Discussion Both number and area of floating leaves indicated the same herbicide effects as wet weight and ash-free dry weight of Potamogeton. Error introduced by the different work steps is small and can be further minimised by a number of method improvements. Recommendations and Perspectives In indoor mesocosm studies, errors due to the perspective adjustment may be circumvented by taking the photos perpendicular to the water surface. Correction for lens aberration, identical light conditions and the use of fluorescence images are considered promising. Field applications are proposed.     

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Background, Aim and Scope Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented for long range transport of these conjugates in plants, rather than storage in the vacuole. To our knowledge this is the first report about the unidirectional long range transport of xenobiotic conjugates in plants and the exudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds which give them similar advantages as animals. Materials and Methods: Barley plants (Hordeum vulgare L. cv. Cherie) were grown in Petri dishes soaked with tap water in the greenhouse. - Fluorescence Microscopy. Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers for our experiments. Their transport in the root could be followed sensitively with very good temporal and spatial resolution. Roots of barley seedlings were cut under water and the end at which xenobiotics were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). - Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. - Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results: The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min-1. The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. Discussion: It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. - On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Conclusions: Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. Recommendations and Perspectives: The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exuded metabolites on the biology of rhizosphere bacteria and fungi.     

Phytoextraction of lead-contaminated soil using vetivergrass (<Emphasis Type="Italic">Vetiveria zizanioides</Emphasis> L.), cogongrass (<Emphasis Type="Italic">Imperata cylindrica</Emphasis> L.) and carabaograss (<Emphasis Type="Italic">Paspalum conjugatum</Emphasis> L.)

Background, Aims and Scope The global problem concerning contamination of the environment as a consequence of human activities is increasing. Most of the environmental contaminants are chemical by-products and heavy metals such as lead (Pb). Lead released into the environment makes its way into the air, soil and water. Lead contributes to a variety of health effects such as decline in mental, cognitive and physical health of the individual. An alternative way of reducing Pb concentration from the soil is through phytoremediation. Phytoremediation is an alternative method that uses plants to clean up a contaminated area. The objectives of this study were: (1) to determine the survival rate and vegetative characteristics of three grass species such as vetivergrass, cogongrass and carabaograss grown in soils with different Pb levels; and (2) to determine and compare the ability of the three grass species as potential phytoremediators in terms of Pb accumulation by plants. Methods The three test plants: vetivergrass (Vetiveria zizanioides L.); cogongrass (Imperata cylindrica L.); and carabaograss (Paspalum conjugatum L.) were grown in individual plastic bags containing soils with 75 mg kg⁻¹ (37.5 kg ha⁻¹) and 150 mg kg⁻¹ (75 kg ha⁻¹) of Pb, respectively. The Pb contents of the test plants and the soil were analyzed before and after experimental treatments using an atomic absorption spectrophotometer. This study was laid out following a 3 × 2 factorial experiment in a completely randomized design. Results On the vegetative characteristics of the test plants, vetivergrass registered the highest whole plant dry matter weight (33.85–39.39 Mg ha⁻¹). Carabaograss had the lowest herbage mass production of 4.12 Mg ha⁻¹ and 5.72 Mg ha⁻¹ from soils added with 75 and 150 mg Pb kg⁻¹, respectively. Vetivergrass also had the highest percent plant survival which meant it best tolerated the Pb contamination in soils. Vetivergrass registered the highest rate of Pb absorption (10.16 ± 2.81 mg kg⁻¹). This was followed by cogongrass (2.34 ± 0.52 mg kg⁻¹) and carabaograss with a mean Pb level of 0.49 ± 0.56 mg kg⁻¹. Levels of Pb among the three grasses (shoots + roots) did not vary significantly with the amount of Pb added (75 and 150 mg kg⁻¹) to the soil. Discussion Vetivergrass yielded the highest biomass; it also has the greatest amount of Pb absorbed (roots + shoots). This can be attributed to the highly extensive root system of vetivergrass with the presence of an enormous amount of root hairs. Extensive root system denotes more contact to nutrients in soils, therefore more likelihood of nutrient absorption and Pb uptake. The efficiency of plants as phytoremediators could be correlated with the plants’ total biomass. This implies that the higher the biomass, the greater the Pb uptake. Plants characteristically exhibit remarkable capacity to absorb what they need and exclude what they do not need. Some plants utilize exclusion mechanisms, where there is a reduced uptake by the roots or a restricted transport of the metals from root to shoots. Combination of high metal accumulation and high biomass production results in the most metal removal from the soil. Conclusions The present study indicated that vetivergrass possessed many beneficial characteristics to uptake Pb from contaminated soil. It was the most tolerant and could grow in soil contaminated with high Pb concentration. Cogongrass and carabaograss are also potential phytoremediators since they can absorb small amount of Pb in soils, although cogongrass is more tolerant to Pb-contaminated soil compared with carabaograss. The important implication of our findings is that vetivergrass can be used for phytoextraction on sites contaminated with high levels of heavy metals; particularly Pb. Recommendations and Perspectives High levels of Pb in localized areas are still a concern especially in urban areas with high levels of traffic, near Pb smelters, battery plants, or industrial facilities that burn fuel ending up in water and soils. The grasses used in the study, and particularly vetivergrass, can be used to phytoremediate urban soil with various contaminations by planting these grasses in lawns and public parks. ESS-Submission Editor: Dr. Willie Peijnenburg (wjgm.peijnenburg@rivm.nl)     

A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

Energy,food, and land — The ecological traps of humankind

Humans’ superiority over all other organisms on earth rests on five main foundations: command of fire requiring fuel; controlled production of food and other biotic substances; utilization of metals and other non-living materials for construction and appliances; technically determined, urban-oriented living standard; economically and culturally regulated societal organization. The young discipline of ecology has revealed that the progress of civilization and technology attained, and being further pursued by humankind, and generally taken for granted and permanent, is leading into ecological traps. This metaphor circumscribes ecological situations where finite resources are being exhausted or rendered non-utilizable without a realistic prospect of restitution. Energy, food and land are the principal, closely interrelated traps; but the absolutely decisive resource in question is land whose increasing scarcity is totally underrated. Land is needed for fulfilling growing food demands, for producing renewable energy in the post-fossil and post-nuclear era, for maintaining other ecosystem services, for urban-industrial uses, transport, material extraction, refuse deposition, but also for leisure, recreation, and nature conservation. All these needs compete for land, food and non-food biomass production moreover for good soils that are scarcer than ever. We are preoccupied with fighting climate change and loss of biodiversity; but these are minor problems we could adapt to, albeit painfully, and their solution will fail if we are caught in the interrelated traps of energy, food, and land scarcity. Land and soils, finite and irreproducible resources, are the key issues we have to devote our work to, based on careful ecological information, planning and design for proper uses and purposes. The article concludes with a short reflection on economy and competition as general driving forces, and on the role and reputation of today’s ecology. Updated version of the keynote lecture presented at the EcoSummit 2007 in Beijing, China, May 24. The article is gratefully dedicated to the memory of my late colleague and friend Frank B. Golley.