Effect of algae growth on aerobic granulation and nutrients removal from synthetic wastewater by using sequencing batch reactors.

The effect of algae growth on aerobic granulation and nutrients removal was studied in two identical sequencing batch reactors (SBRs). Sunlight exposure promoted the growth of algae in the SBR (Rs), forming an algal-bacterial symbiosis in aerobic granules. Compared to the control SBR (Rc), Rs had a slower granulation process with granules of loose structure and smaller particle size. Moreover, the specific oxygen uptake rate was significantly decreased for the granules from Rs with secretion of 25.7% and 22.5% less proteins and polysaccharides respectively in the extracellular polymeric substances. Although little impact was observed on chemical oxygen demand (COD) removal, algal-bacterial symbiosis deteriorated N and P removals, about 40.7-45.4% of total N and 44% of total P in Rs in contrast to 52.9-58.3% of TN and 90% of TP in Rc, respectively. In addition, the growth of algae altered the microbial community in Rs, especially unfavorable for Nitrospiraceae and Nitrosomonadaceae.

Differences in nutrient uptake capacity of the benthic filamentous algae Cladophora sp., Klebsormidium sp. and Pseudanabaena sp. under varying N/P conditions.

The N/P ratio of wastewater can vary greatly and directly affect algal growth and nutrient removal process. Three benthic filamentous algae species Cladophora sp., Klebsormidium sp. and Pseudanabaena sp. were isolated from a periphyton bioreactor and cultured under laboratory conditions on varying N/P ratios to determine their ability to remove nitrate and phosphorus. The N/P ratio significantly influenced the algal growth and phosphorus uptake process. Appropriate N/P ratios for nitrogen and phosphorus removal were 5-15, 7-10 and 7-20 for Cladophora sp., Klebsormidium sp. and Pseudanabaena sp., respectively. Within these respective ranges, Cladophora sp. had the highest biomass production, while Pseudanabaena sp. had the highest nitrogen and phosphorus contents. This study indicated that Cladophora sp. had a high capacity of removing phosphorus from wastewaters of low N/P ratio, and Pseudanabaena sp. was highly suitable for removing nitrogen from wastewaters with high N/P ratio.

Highly-efficient enzymatic conversion of crude algal oils into biodiesel.

Energy-intensive chemical conversion of crude algal oils into biodiesel is a major barrier for cost-effective algal biofuel production. To overcome this problem, we developed an enzyme-based platform for conversion of crude algal oils into fatty acid methyl esters. Crude algal oils were extracted from the oleaginous microalga Nannochloropsis oceanica IMET1 and converted by an immobilized lipase from Candida antarctica. The effects of different acyl acceptors, t-butanol as a co-solvent, oil to t-butanol ratio, oil to methanol ratio, temperature and reaction time on biodiesel conversion efficiency were studied. The conversion efficiency reached 99.1% when the conversion conditions were optimized, i.e., an oil to t-butanol weight ratio of 1:1, an oil to methanol molar ratio of 1:12, and a reaction time of 4h at 25°C. The enzymatic conversion process developed in this study may hold a promise for low energy consumption, low wastewater-discharge biochemical conversion of algal feedstocks into biofuels.

Moderate changes in nutrient input alter tropical microbial and protist communities and belowground linkages.

We investigated the response of soil microbial communities in tropical ecosystems to increased nutrient deposition, such as predicted by anthropogenic change scenarios. Moderate amounts of nitrogen and phosphorus and their combination were added along an altitudinal transect. We expected microorganisms and microbial grazers (testate amoebae) to significantly respond to nutrient additions with the effect increasing with increasing altitude and with duration of nutrient additions. Further, we expected nutrients to alter grazer-prey interrelationships. Indeed, nutrient additions strongly altered microbial biomass (MB) and community structure as well as the community structure of testate amoebae. The response of microorganisms varied with both altitude and duration of nutrient addition. The results indicate that microorganisms are generally limited by N, but saprotrophic fungi also by P. Also, arbuscular mycorrhizal fungi benefited from N and/or P addition. Parallel to MB, testate amoebae benefited from the addition of N but were detrimentally affected by P, with the addition of P negating the positive effect of N. Our data suggests that testate amoeba communities are predominantly structured by abiotic factors and by antagonistic interactions with other microorganisms, in particular mycorrhizal fungi, rather than by the availability of prey. Overall, the results suggest that the decomposer system of tropical montane rainforests significantly responds to even moderate changes in nutrient inputs with the potential to cause major ramifications of the whole ecosystem including litter decomposition and plant growth.

Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater.

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

Restoration of taxonomic and functional genes after bioaugmentation of petroleum contaminated soil.

Soil microbial ecosystems are responsive to environmental changes that underpin the biological functions of the soil. The present study was conducted to profile variations in the microbial ecological system of remediated soil (R) and petroleum contaminated soil (P) based on comparisons with soil that had not been contaminated (N), using a cloning library of taxonomic genes (16S rRNA gene for bacteria and 18S rRNA gene for eukaryotes) and functional genes (nifH, amoA and narG). The results showed that N and R had a similar distribution in both the taxonomic genes and functional genes for bacteria and eukaryotes, which were dominated by Proteobacteria and Arthropoda, respectively. Phylogenetic analysis based on the nifH gene showed that the sequences from the three soils were clustered into six taxonomic groups, Actinobacteridae, and Alpha-, Beta-, Gamma- and Delta-proteobacteria, as well as an unclassified group. Evaluation of the amoA gene revealed that all sequences derived from the three samples belonged to Betaproteobacteria. The R and N soil had similar Shannon-Wiener diversity index (H') values, both of which were significantly higher than that of the P soil. The most abundant bacterial phylotype identified in the N and R soils were the same and were related to an uncultured bacterial clone (GAN-SB17, FN423475). None of the narG genes were found in the P soil. Similar results in terms of distribution, composition and the related index were obtained for nifH and amoA. These parameters may comprise a biological ecology index that may be applied to aid the design, implementation and evaluation of soil bioremediation.

Nutrient removal in wastewater treatment high rate algal ponds with carbon dioxide addition.

The influence of CO2 addition to high rate algal ponds (HRAPS) on nitrogen removal was investigated using two pilot-scale HRAPs operated with different hydraulic retention times (HRT: 4 and 8 days), and was compared to the nitrogen removal by the 8-day HRT pond before CO2 addition was installed. Nitrogen balances were calculated by partitioning total nitrogen into organic and inorganic nitrogen (NH4+-N and NO3--N), and by separation of the organic nitrogen into particulate (PON) and dissolved organic nitrogen (DON). PON was further divided into algal organic nitrogen (AON) and bacteria organic nitrogen (BON) to investigate nitrogen mass flow in the HRAPS. This research shows that the proportion of algae in the algal/bacterial biomass in the longer 8-day HRT HRAP8d (55.6%) was appreciably lower than that in the shorter 4-day HRT HRAP4d (80.5%) when CO2 was added to control the maximum pH to <8.0 during the summer. Higher bacterial biomass in the longer 8-day HRT HRAP corresponded with higher nitrification rates, indicating that the longer 8-day HRT in the summer was detrimental for two reasons: lower algal productivity and increased nitrogen loss through nitrification/denitrification. Overall nitrogen removal of approximately 60% in the HRAPS with CO2 addition was mainly achieved by algal assimilation followed by sedimentation in the settling unit.

Effects of total resources, resource ratios, and species richness on algal productivity and evenness at both metacommunity and local scales.

The study of the interrelationship between productivity and biodiversity is a major research field in ecology. Theory predicts that if essential resources are heterogeneously distributed across a metacommunity, single species may dominate productivity in individual metacommunity patches, but a mixture of species will maximize productivity across the whole metacommunity. It also predicts that a balanced supply of resources within local patches should favor species coexistence, whereas resource imbalance would favor the dominance of one species. We performed an experiment with five freshwater algal species to study the effects of total supply of resources, their ratios, and species richness on biovolume production and evenness at the scale of both local patches and metacommunities. Generally, algal biovolume increased, whereas algal resource use efficiency (RUE) and evenness decreased with increasing total supply of resources in mixed communities containing all five species. In contrast to predictions for biovolume production, the species mixtures did not outperform all monocultures at the scale of metacommunities. In other words, we observed no general transgressive overyielding. However, RUE was always higher in mixtures than predicted from monocultures, and analyses indicate that resource partitioning or facilitation in mixtures resulted in higher-than-expected productivity at high resource supply. Contrasting our predictions for the local scale, balanced supply of resources did not generally favor higher local evenness, however lowest evenness was confined to patches with the most imbalanced supply. Thus, our study provides mixed support for recent theoretical advancements to understand biodiversity-productivity relationships.

Rapid shifts in the structure and composition of a protistan assemblage during bottle incubations affect estimates of total protistan species richness.

Changes in the structure and composition of a protistan community were characterized through the analysis of small-subunit ribosomal RNA gene (18S) sequences for a 3-day bottle incubation using a single sample collected in the western North Atlantic. Cloning and sequencing was used to investigate changes in perceived species richness and diversity as a consequence of environmental perturbation. The treatments included a control (unamended seawater), inorganic nutrient enrichment, and enrichment with a complex organic mixture. Five clone libraries were constructed and analyzed at the time of collection (t-0 h) and after 24 (t-24 h) and 72 (t-72 h) h for the control, and at t-72 h for the inorganic and organic enrichments, resulting in an analysis of 1,626 partial 18S rDNA sequences that clustered into 238 operational taxonomic units (OTUs). Analysis of the clone libraries revealed that protistan assemblages were highly dynamic and changed substantially at both the OTU level and higher taxonomic classifications during time frames consistent with many oceanographic methods used for measuring biological rates. Changes were most dramatic in enrichments, which yielded community compositions that were strongly dominated by one or a few taxa. Changes in community structure during incubation dramatically influenced estimates of species richness, which were substantially lower with longer incubation and especially with amendment, even though all incubated samples originated from the same aliquot of seawater. Containment and enrichment of the seawater sample led to the detection of otherwise undetected protistan taxa, suggesting that characterization of protistan diversity in a sample only at the time of collection could lead to an underrepresentation of unique taxa. Additionally, the rapid increase in the relative abundance of some members of the "rare biosphere" in our results implies an ecological importance of at least some of the taxa comprising the "rare biosphere."

Meta-analyses of effects of phytochemicals on digestibility and rumen fermentation characteristics associated with methanogenesis.

BACKGROUND: A meta-analysis study was conducted to investigate the changes in rumen fermentation characteristics when methane inhibition by phytochemicals is employed. The whole database containing 185 treatment means from 36 published studies was divided into four subsets according to the major phytochemicals used in the studies, i.e. saponins, tannins, essential oils (EO) and organosulfur compounds (OS). RESULTS: Changes in protozoal numbers showed linear relationships with changes in methane production by saponins (R(2) = 0.48), tannins (R(2) = 0.30) and EO (R(2) = 0.20) but not OS. Concentrations of total volatile fatty acids (VFA) and acetate did not show any relationship (P > 0.1) with changes in methane due to saponins. However, propionate production increased linearly with increasing inhibition of methane (R(2) = 0.31), which resulted in a linear (R(2) = 0.26) decrease in acetate/propionate ratio (A/P) with decreasing methane production. Concentrations of total VFA, acetate and propionate did not change with changes in methane production by tannins. However, A/P showed a significant linear relationship (R(2) = 0.27) with decreasing methane formation. Concentrations of total VFA (R(2) = 0.44) and propionate (R(2) = 0.15) changed linearly and positively with changes in methane production by EO. However, acetate production (R(2) = 0.22) and A/P (R(2) = 0.17) increased linearly with increasing inhibition of methane by EO. Changes in concentrations of total VFA (R(2) = 0.60) and acetate (R(2) = 0.35) decreased linearly while those of propionate increased linearly (R(2) = 0.23) with increasing inhibition of methane by OS. Consequently, A/P decreased linearly (R(2) = 0.30) with decreasing methane production by OS. Digestibilities of organic matter (OM) and neutral detergent fibre were not affected by inhibition of methane production by saponins, EO and OS, but digestibility of OM decreased with decreasing methane production by tannins. CONCLUSION: The inhibition of methane production by phytochemicals results in changes in rumen fermentation that differ depending on the types of phytochemicals.

Balancing between retention and flushing in river networks--optimizing nutrient management to improve trophic state.

River basin management can frequently involve decisive situations, when conflicting interests must be resolved. In the Zala River catchment (Western Hungary) local efforts to improve water quality by reducing algal biomass are not always harmonized with the requirement of sustaining the same objective in its recipient, Lake Balaton. The PhosFate catchment model is a GIS tool designed to estimate the spatial variability and fate of diffuse phosphorus emission during transport. Besides diffuse pollution, a simplified annual hydrologic balance is also calculated. A new module was added to PhosFate that tracked the development of entrained algae during their travel downstream. The extended model was used to simulate the current average algal concentrations in the river network. The numerous small reservoirs and impoundments on the tributaries of the Zala River were identified as the key elements in determining algal biomass, since they fundamentally increase the water residence time (WRT) in the system. Without reservoirs, the short WRT in the drainage network would successfully prevent the development of suspended algal biomass despite the fairly high SRP concentrations. However, the removal of such standing waters is impossible for socio-economic reasons and reducing the overall P load to Lake Balaton would also require increasing WRT in the system. As a resolution to these conflicting interests, a hybrid management strategy was designed to simultaneously reach both goals: (i) switching from WRT to P limitation in reservoirs responsible for most of algal growth, and (ii) optimized deployment of buffer zones and the introduction of best agricultural practices on the remaining majority of the catchment to reduce the overall P load. The suggested management approach could be applied in other river catchments too, due to the extensive presence of reservoirs and impoundments in many stream networks.

Numerical investigation of nutrient limitations in the Bohai Sea.

River discharges are the important freshwater and nutrient sources for Bohai Sea (BS), and have a profound impact on the local marine environment. In this paper, the annual cycles of nutrient and phytoplankton dynamics in 1980s were reproduced using a coupled biogeochemical-physical model. Based on the validated simulations, the nutrient limitation characters were further investigated by running the model with the riverine nutrient altered, first enriching nitrogen and then phosphorus. It was found that although the riverine N:P ratios in Yellow and Haihe Rivers were much higher than the Redfield number, the nitrogen enrichment was still able to enhance the algae bloom in Laizhou and Bohai Bays. On the other hand, the response of algae growth to phosphorus enrichment was not thus obvious, which suggests that the local phytoplankton dynamics was characterized by the nitrogen limitation. Simulations also show that the nitrogen enrichment is generally accompanied by the phosphorus consumption, so a shift from nitrogen limitation to phosphorus limitation may occur if such a trend continues.

River phosphorus cycling: separating biotic and abiotic uptake during short-term changes in sewage effluent loading.

Medium to small scale point sources continue to threaten river ecosystems through P loadings. The capacity and timescales of within-river processing and P retention are a major factor in how rivers respond to, and protect downstream ecosystems from, elevated concentrations of soluble reactive P (SRP). In this study, the bio-geochemical response of a small river (approximately 40 km(2) catchment area) was determined before, during and after exposure to a fourteen day pulse of treated sewage effluent using an upstream reach as a control. A wide array of approaches (batch and column simulations to in-situ whole stream metabolism) allowed independent comparison and quantification, of the relative contribution of abiotic and biotic processes in-river P cycling. This enabled, for the first time, separating the relative contributions of algae, bacteria and abiotic sorption without the use of labelled P (radioisotope). An SRP mass balance showed that the ecosystem switched from a P sink (during effluent inputs) to a P source (when effluent flow ceased). However, 65-70% of SRP was retained during the exposure time and remained sequestered two-weeks after-effluent flow ceased. Batch studies treated with biocide gave unrealistic results, but P uptake rates derived by other methods were highly comparable. Downstream of the effluent input, net P uptake by algae, bacteria and sediment (including the biofilm polysaccharide matrix) were 0.2 (+/-0.1), 0.4 (+/-0.3), and 1.0 (+/-0.9) mmol m(-2) day(-1) during effluent exposure. While autotrophic production did not respond to the effluent exposure, heterotrophic production increased by 67% relative to the control and this translated into a 50% increase in biological P uptake rate. Therefore, both biological and abiotic components of stream ecosystems uptake P during exposure to treated sewage effluent P inputs, and maintain a long 'memory' of this input in terms of P storage for considerable timescales after loading.

A cost/effective screening method for assessing the toxicity of nutrient rich effluents to algae.

Screening whole effluent toxicity tests are cost/effective methods for detecting the presence of toxic concentrations of unknown pollutants, but the application must solve the problem associated with the effect of high and variable concentrations of nutrients in the effluent on the results of algal toxicity tests. This work proposes a cost/effective test, based on three dilution levels measured at a single point time and a discriminant model for establishing if this kind of complex samples, with difficult interpretation of dilution-response curves, should be considered toxic to algae. This procedure identified properly around 85% of the samples considered toxic by expert judgement.

Modeling continuous cultures of microalgae colimited by nitrogen and phosphorus.

It is well documented that the combination of low nitrogen and phosphorus resources can lead to situations where colimitation of phytoplankton growth arises, yet the underlying mechanisms are not fully understood. Here, we propose a Droop-based model built on the idea that colimitation by nitrogen and phosphorus arises from the uptake of nitrogen. Indeed, since N-porters are active systems, they require energy that could be related to the phosphorus status of the cell. Therefore, we assumed that N uptake is enhanced by the P quota. Our model also accounts for the biological observations that uptake of a nutrient can be down-regulated by its own internal quota, and succeeds in describing the strong contrast for the non-limiting quotas under N-limited and P-limited conditions that was observed on continuous cultures with Selenastrum minutum and with Isochrysis affinis galbana. Our analysis suggests that, regarding the colimitation concept, N and P would be better considered as biochemically dependent rather than biochemically independent nutrients.

Methyl jasmonate- or gibberellins A3-induced astaxanthin accumulation is associated with up-regulation of transcription of beta-carotene ketolase genes (bkts) in microalga Haematococcus pluvialis.

The microalga Haematococcus pluvialis accumulates astaxanthin in response to abiotic stresses. Since methyl jasmonate (MJ) and gibberellins A(3) (GA(3)) are involved in the stress responses of plants, the impact of these compounds on astaxanthin metabolism was studied. Alga cells treated separately with MJ and GA(3) accumulated more astaxanthin than the controls. MJ and GA(3) treatment increased the transcription of three beta-carotene ketolase genes (bkts). MJ- and GA(3)-responsive cis-acting elements were identified in the 5'-flanking regions of bkt genes. These results suggest that MJ and GA(3) constitute molecular signals in the network of astaxanthin accumulation. Induction of astaxanthin accumulation by MJ or GA(3) without any other stimuli presents an attractive application potential.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae.

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

The effects of top-down versus bottom-up control on benthic coral reef community structure.

While climate change and associated increases in sea surface temperature and ocean acidification, are among the most important global stressors to coral reefs, overfishing and nutrient pollution are among the most significant local threats. Here we examined the independent and interactive effects of reduced grazing pressure and nutrient enrichment using settlement tiles on a coral-dominated reef via long-term manipulative experimentation. We found that unique assemblages developed in each treatment combination confirming that both nutrients and herbivores are important drivers of reef community structure. When herbivores were removed, fleshy algae dominated, while crustose coralline algae (CCA) and coral were more abundant when herbivores were present. The effects of fertilization varied depending on herbivore treatment; without herbivores fleshy algae increased in abundance and with herbivores, CCA increased. Coral recruits only persisted in treatments exposed to grazers. Herbivore removal resulted in rapid changes in community structure while there was a lag in response to fertilization. Lastly, re-exposure of communities to natural herbivore populations caused reversals in benthic community trajectories but the effects of fertilization remained for at least 2 months. These results suggest that increasing herbivore populations on degraded reefs may be an effective strategy for restoring ecosystem structure and function and in reversing coral-algal phase-shifts but that this strategy may be most effective in the absence of other confounding disturbances such as nutrient pollution.

New evidences of Roundup (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems.

Argentina is the second largest world producer of soybeans (after the USA) and along with the increase in planted surface and production in the country, glyphosate consumption has grown in the same way. We investigated the effects of Roundup (glyphosate formulation) on the periphyton colonization. The experiment was carried out over 42 days in ten outdoor mesocosms of different typology: "clear" waters with aquatic macrophytes and/or metaphyton and "turbid" waters with great occurrence of phytoplankton or suspended inorganic matter. The herbicide was added at 8 mg L(-1) of the active ingredient (glyphosate) in five mesocosms while five were left as controls (without Roundup addition). The estimate of the dissipation rate (k) of glyphosate showed a half-life value of 4.2 days. Total phosphorus significantly increased in treated mesocosms due to Roundup degradation what favored eutrophication process. Roundup produced a clear delay in periphytic colonization in treated mesocosms and values of the periphytic mass variables (dry weight, ash-free dry weight and chlorophyll a) were always higher in control mesocosms. Despite the mortality of algae, mainly diatoms, cyanobacteria was favored in treated mesocosms. It was observed that glyphosate produced a long term shift in the typology of mesocosms, "clear" turning to "turbid", which is consistent with the regional trend in shallow lakes in the Pampa plain of Argentina. Based on our findings it is clear that agricultural practices that involve the use of herbicides such as Roundup affect non-target organisms and the water quality, modifying the structure and functionality of freshwater ecosystems.