Bacterial communities in water and sediment shaped by paper mill pollution and indicated bacterial taxa in sediment in Daling River

Effluents from paper mills are highly toxic and are a major source of aquatic pollution. In this study, we collected water and sediment samples to examine the microbial communities using denaturing gradient gel electrophoresis and identified bacterial taxa greatly affected by paper mill pollution using next-generation sequencing data. Our results indicated that bacterial communities in downstream sediments were similar to those in paper mill discharge sites, indicating obvious effects of pollution, while bacterial communities in downstream water samples showed similar profiles to those in upstream sites, both being quite different from the bacterial communities in paper mill discharge sites. This was possibly because of the short contact period. In addition, bacterial communities in the estuary were quite different from those in other water and sediment samples, which was owing to the special habitat type. Considering the storage of paper mill pollutants in sediment and the significant effect on shifts in bacterial communities, we selected Clostridia and Epsilonproteobacteria at the class level and Fusibacter and Desulfobulbus at the genus level as bacterial indicators of paper mill pollution. To monitor the remediation of polluted aquatic environments, we propose Sphingobacteria, Alphaproteobacteria, Actinobacteria, Subdivision3, Planctomycetacia and Verrucomicrobiae at the class level and Bacillus, Steroidobacter, Nocardioides, Terrimonas, Pirellula and Methylibium at the genus level.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina.

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

Effects of cooling water discharge on the structure and dynamics of epilithic algal communities in the northern Baltic

The Forsmark Biotest Basin is a shallow coastal ecosystem that receives brackish cooling-water discharge from a nuclear power plant. The effects of the discharge on epilithic algal communities were investigated by analysing samples taken every third week throughout one year at 11 sites differentially affected by temperature and/or flow rate enhancement. Community variation was summarized in a canonical correspondence analysis (CCA) of species abundances as a function of site and date. The temperature increase favoured blue-green algae at the expense of red and brown algae. Blue-green algae were however abundant in summer in stagnant water, whether heated or not, and some red and brown algae became abundant in winter in heated sites with flowing water. Green algae and diatoms increased in biomass in the heated sites, but not in relative cover-abundance. The absence of ice and snow cover at sites with heated and/or flowing water caused autumn species to persist into winter, because of the higher light intensity (compared with natural conditions) and the absence of the mechanical abrasion by ice. The thermal discharge lowered species diversity (Shannon-Weaver index) both in summer and winter at sites with flowing water, but not at sites with quiescent or stagnant water. CCA showed alternate periods of stability and rapid change within the seasonal cycle. Individual species were placed according to their optimum; red and brown algae in winter/spring, green algae in spring/summer, blue-green algae in summer, and diatoms at various times. Exceptions to this pattern were species endo- or epiphytic on species of a different group. Analysis of the effects of temperature, flow rate and ice cover on the seasonal pattern of particular species showed that different species respond in individualistic ways to different combinations of these environmental variables.     

Induction of micronucleus and nuclear alterations in fish (Oreochromis niloticus) by a petroleum refinery effluent

In this study, micronucleus and nuclear alterations tests were performed on erythrocytes of Oreochromis niloticus (Perciformes, Cichlidae) in order to evaluate the water quality of the Atibaia river, in an area that receives effluents discharge of a petroleum refinery and also to evaluate the effectiveness of the treatments used by the refinery. Water samples were collected in five different sites related with a refinery from S?o Paulo State, Brazil. For the micronucleus and nuclear alterations tests, O. niloticus specimens were exposed for 72 h to the water samples and in pure ground water (negative control). The results herein obtained indicated that the treatments used by the refinery diminished the cytogenetic damage; however they were not fully effective, since the final mill has induced damages in the genetic material of the test organism.     

Bacterial community shift revealed Chromatiaceae and Alcaligenaceae as potential bioindicators in the receiving river due to palm oil mill effluent final discharge

A thorough outlook on the effect of palm oil mill effluent (POME) final discharge towards bacterial community dynamics in the receiving river is provided in this study by using a high-throughput MiSeq. The shift of bacterial composition could be used to determine the potential bacterial indicators to indicate contamination caused by POME. This study showed that the POME final discharge did not only alter the natural physicochemical properties of the river water but also caused the reduction of bacterial diversity in the receiving river. The Chromatiaceae and Alcaligenaceae which were not detected in the upstream but were detected in the downstream part of the river are proposed as the indicator bacteria to indicate the river water contamination caused by POME final discharge. The emergence of either one or both bacteria in the downstream part of the river were shown to be carried over by the effluent. Therefore, an accurate pollution monitoring approach using bacterial indicator is expected to complement the conventional POME pollution assessment method which is currently dependent on the physicochemical properties of the final discharge. This is the first study that reported on the potential indicator bacteria for the assessment of river water contamination caused by POME final discharge.     

Environmental factors which influence the sink of silica in the limnetic system of the large monomictic Lake Biwa and its watershed in Japan

Dissolved silica (DSi) and its associated biological and physicochemical factors were measured in Lake Biwa, Japan and its watershed from 2002 to 2003 in order to clarify seasonal variations in the magnitude of the sink of silica and the factors that influence it within the limnetic system. Consequently, it is concluded that Lake Biwa is a noticeable body of water where a massive sink of silica is caused. Calculated silica sedimentation in Lake Biwa was 2.0 × 10⁷ kg Si year⁻¹ (7.1 × 10⁸ mol Si year⁻¹) which is equivalent to about 80% of the annual inflow discharge of DSi to Lake Biwa. The magnitude of the sink varies seasonally by increasing in the winter holomictic stirring period, since it is greatly affected by the species composition of phytoplankton, the load of phosphorus and the condition of stratification. It seems reasonable to suppose that the DSi in Lake Biwa is removed mainly by biological processes, i.e., the assimilation of DSi by large centric diatoms and its accumulation in their frustules. Such silica sinks occur naturally in deeper stagnant waters, providing extended water residence time and supplying a certain amount of nutrients. These findings indicate that an increase in nutrient loads and abundance of stagnant water due to the construction of large dams lead to an expansion in the magnitude of the silica sink in a limnetic system.     

Source and extent of Klebsiella pneumoniae in the paper industry.

Three pulp and paper mill processing plants were evaluated for fecal coliform and Klebsiella pneumoniae bacterial concentrations. Freshwater consumed by paper industries contained minimum detectable levels of K. pneumoniae, less than 10 organisms per 100 ml. Elevated concentrations of K. pneumoniae could be traced from early pulping stages to water processing reuse systems. Concentrations of K. pneumoniae (thermotolerant and thermointolerant) ranged from 40,000 organisms per 100 ml to an estimated 3 x 10(6) organisms per 100 ml. K. pneumoniae biotyping provided evidence for the selective growth and persistence of K. pneumoniae from the initial wood washing stages through to the final effluent discharge. Wastewater treatment had limited effects in reducing K. pneumoniae concentrations. K. pneumoniae levels ranged from 40 organisms per 100 ml to an estimated 10(6) organisms per 100 ml. The presence of K. pneumoniae in water indicates degraded water quality, and its significance with regard to human health effects has yet to be examined.     

Pulp and paper mill waste pollution in the Swale,a tidal channel on the east coast of England

The effect of pulp and paper mill waste discharges in the Swale, a tidal channel on the southeast coast of England, has been studied. A pulsing tidal movement in the Swale results in effluent being trapped within the estuary for up to 20 days. This has resulted in adverse effects on the quality of the water and sediment along a substantial part of the estuary with the greatest effect occurring east of the mill in the direction of the residual current. A reduction in the dissolved oxygen concentration occurred along a 13-km stretch of the estuary with mean values falling to 53 % saturation near the mill. High levels of organic matter in the sediment reflect the deposition of material from the mill effluent stream and loss on ignition values of up to 12 % were found east of the mill, falling to 5 % within 4 km northwest of the mill. The macrofauna of the intertidal mudflats was typical of a stressed environment with a low number of species and with a few being represented by large numbers of individuals. The dominant species was the sabellid polychaeteManayunkia aestuarina which reached peak numbers of over 1.5 million m⁻², 1.5 km east of the mill. An increase in the total biomass near the mill is thought to be associated with organic enrichment from the mill wastes.     

Impacts of deep open drains on water quality and biodiversity of receiving waterways in the Wheatbelt of Western Australia

Extensive networks of deep drains are being built in Western Australia to reduce the effects of dryland salinity on agricultural lands. Most of these drains discharge into natural river and wetland systems, with little consideration given to the environmental impacts. This study examined the downstream ecological impacts of one of the oldest deep drain networks in Western Australia, located in the Wakeman subcatchment near Narembeen. Twelve sites were sampled bi-monthly from October 2004 to September 2006. On each occasion, water quality parameters were measured and the macro-invertebrate fauna was sampled. Significant differences in water quality and macro-invertebrates were observed between the untreated sites and those affected by the drain discharge. Surface water at untreated sites was always fresh (<3 ppt), alkaline (pH 7.6–8.9) and turbid (49–600 NTU), whereas treatment sites were always saline (28–147 ppt), acidic (pH 1.9–3.8) and mostly clear (0–100 NTU). No recovery of water quality was observed with distance from discharge point (20 km). Invertebrates reflected differences in water quality, with drain discharge resulting in a sharp decline in species richness, and significant changes in macro-invertebrate community composition. Sites affected by drain discharge were dominated by fly larvae such as Orthocladiinae and Ceratopogonidae. Microcrustaceans were far more abundant at sites unaffected by drainage. The ecological values of Wheatbelt streams are likely to be further compromised by discharge of poor water quality from deep drainage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens     

Northern pike (Esox lucius L.) and aquatic vegetation,tools in the management of fisheries and water quality in shallow waters

Species and size composition of fish communities in shallow stagnant waters appear to be associated with the type, abundance and pattern of the vegetation. Man-induced impacts as eutrophication, and suppression of vegetation for reasons of water quantity management or angling pleasure may induce irreversible changes in the aquatic ecosystem. Water quality management should aim at restoring former pike habitat. Submerged weeds are important to that effect. These habitats are characterized by relatively low densities of fish stocks. Stocking of bream and carp interferes strongly with these objectives. With respect to fish stocks, interests of water quality and fishery management may be opposed.     

Thermal stress markers in <Emphasis Type="Italic">Colpophyllia</Emphasis><Emphasis Type="Italic">natans</Emphasis> provide an archive of site-specific bleaching events

Tropical coral reef monitoring relies heavily on in situ diver observations. However, in many reef regions resources are not available to regularly monitor reefs. This lack of historical baseline data makes it difficult to determine how different reefs respond to environmental stressors and what the implications are for management. To test whether coral cores could be used to identify bleaching events retrospectively, three sites in Tobago with pre-existing reef data including water quality and bleaching observations were identified. Colpophyllia natans cores were examined for growth anomalies which occurred during periods of thermal stress. If present, anomalies were compared to in situ, real-time bleaching observations and water quality data. Interestingly, sites with better water quality during the 2005 thermal anomaly were less prone to bleaching. We suggest that by reducing terrestrial run-off (e.g., sediment and nutrients), and therefore improving marine water quality, reef managers could enhance near-shore coral reef resilience during high-temperature events.     

The impact of effluents of Pitkäranta pulp mill on the water quality of Lake Ladoga: a model study

The aim of this study is to show the effects of the Pitkäranta pulp mill on the water quality of Northern Ladoga by using water quality models. The effluent loading of the pulp mill with its full production capacity is as follows: Water flow 85 000 m³ d^–1 BOD₅ 2.4 t d^–1 Suspended solids 4.1 t d^–1 tot-N 330 kg d^–1 tot-P 68 kg d^–1 COD(Cr) 14.4 t d^–1 Org. C 6 t d^–1 Lignosulphonates 9.4 t d^–1 Loadings of lignosulphonates and organic C are estimations. Lignosulphonate concentrations of 10.5 mg 1^–1 have been reported in the region of Pitkäranta. The study area of northern Ladoga near the pulp mill was divided into three zones (I, II and III). The mean depth of each of them is considered as 10 m, their respective areas 5, 10 and 50 km², and volumes 50, 100 and 500 million m³. The estimated discharges of the zones are 10, 20 and 50 m³ s^–1, respectively. With the aid of simple water quality models the effects of the pulp mill effluents on the concentrations of oxygen, total phosphorus, lignin, COD(Mn) and Secchi disk depths in each of the zones were estimated. Estimations were made during full production capacity and half production capacity of the pulp mill. The modelling results were compared with the preliminary water analysis results of the Finnish-Russian joint research expedition into Lake Ladoga in August 1993. The results show that near the pulp mill (zone I) phosphorus concentrations are high even with half production capacity. Also lignin and COD(Mn) contents have increased, and oxygen concentrations are low both in the summer and during wintertime. Farther away in the open water (zone III) the pollution effects are low. Eutrophication, indicated by high total phosphorus concentrations, is the main effect of the pulp mill effluents.     

Relationship between the dough quality and content of specific glutenin proteins in wheat mill streams, and its application to making flour suitable for instant Chinese noodles

The content of specific proteins such as high-molecular-weight glutenin subunits HMW-GS 5+10 and low-molecular-weight glutenin subunits LMW-GS KS2 in wheat mill streams of extra-strong Kachikei 33 wheat was quantified by SDS-PAGE and 2D-PAGE. The mill streams showed varied quantities of HMW-GS 5+10 (0.077 to 2.007 mg/g of mill stream), LMW-GS KS2 (0.018 to 0.586 mg/g of mill stream) and total protein (9.42% to 18.98%). The contents of these specific proteins in the mill streams were significantly correlated with the SDS sedimentation volume and the mixing properties, which are respective indices of specific loaf volume and dough strength. The contents of these specific glutenin proteins in the mill streams were therefore found to be significantly important for improving the dough quality suitable for bread and Chinese noodles. Accordingly, we present here the application of this information to the development of an effective method for producing mill streams with high quality and yield that are suitable for instant Chinese noodles.     

Changes in water quality in Lake Päijänne following decrease of effluent load from the pulp and paper industry in 1969–1989

Lake Päijänne, the second largest lake in Finland, has been seriously polluted since the 1960s due to the effluents from pulp and paper mills, notably the Äänekoski sulphate and sulphite pulp mills situated about 50 km north of the lake, and the sulphite pulp mill and paper mills of Jämsänkoski and Kaipola on Central Lake Päijänne.A sulphite lye evaporation and combustion plant installed at the Jämsänkoski sulphite pulp mill in 1969 reduced the organic pollution of Central Päijänne. Lignin concentration decreased and the oxygen balance improved. The sulphite pulp mill was closed in 1981 and replaced by a thermo-mechanical pulp mill. Eutrophication is the main threat to water quality in Central Lake Päijänne at present.A significant improvement in the water quality, especially in oxygen balance, was achieved in the watercourse of Äänekoski and in Northern Päijänne after replacement of the old sulphite and sulphate pulp mills at Äänekoski by a large sulphate pulp mill with a biological purification plant employing the activated sludge method. The BOD₇-loading dropped from 46 to 3–4 t d^–1, but the nutrient loading has not decreased sufficiently, and the Äänekoski watercourse and Northern Päijänne are still eutrophic.     

Prokaryotic microbiota of recycled paper mills with low or zero effluent

The tendency in the paper industry is to close all water loops to save water. This leads to higher process temperatures and may increase the colloidal and dissolved material in the process circulation. Increase of nutrients in the water circuits may favor microbiological growth and fouling. In this paper the chemical and microbial compositions of water circuits and deposits were studied of two closed cycled paper/board mills, one mill totally closed (0 m(3) waste water t(-1)), and the other low discharging (about 4 m(3 )t(-1)). The zero discharge mill accumulated high amounts (>10 g C L(-1)) of organic carbon in the circulation waters, about 40% of which composed of volatile acids (lactic, acetic, propionic and butyric acid). Water contents of sulfate, chloride, sodium and calcium increased to >1 g L(-1) of each. q-PCR targeted on 16S rRNA genes indicated that the bacteria in water circuits were mainly viable cells. In both mills anaerobic growth (10(6)-10(8) CFU mL(-1)) equalled or exceeded aerobic growth, with odor problem but no actual slime problem. The major part (40%) of all identifiable bacterial sequences were closest but yet distant (<96%) to Enterococcus cecorum and in the 4 m(3 )t(-1) discharging mill also Bacillus thermoamylovorans and Bacillus coagulans. Slimes and deposits from the mills contained high amounts, >/=10(8) g(-1), of archaean, but only the genus Methanothrix was identifiable from the cloned sequences. The findings indicate that closing the water circuits strongly limited diversity of the microbiota but allowed efficient mineralization of the dissolved and suspended matter.     

不同流速对流水槽大口黑鲈生长性能、抗氧化能力、能量代谢及组织结构的影响

研究旨在探明池塘内循环流水养殖(Inner-Pond Raceway Aquaculture, IPRA)系统中不同水流速度对大口黑鲈(Micropterus salmoides)生长性能、抗氧化能力、能量代谢及组织结构的影响。选取已驯食好、体质健康、大小均一的大口黑鲈[放养规格为(8.13±0.82) g]192尾,随机分配到4个模拟水槽中,每组3个重复,每个重复16尾。养殖试验设置空白静水组和3个水流速度组,分别为低流速0.2 m/s、中流速0.4 m/s和高流速0.6 m/s,养殖周期为154d。结果显示:(1)中流速组大口黑鲈的增重率和特定生长率显著高于其他3个处理组(P<0.05);中、高流速组肥满度显著低于静水组和低流速组(P>0.05);大口黑鲈内脏脂肪指数随着流速的增加而显著降低(P<0.05);(2)中流速组肌肉粗蛋白显著高于其他3个处理组(P<0.05),静水组和低流速组肌肉粗脂肪含量显著高于中、高流速组(P<0.05);中、高流速组肝脏粗蛋白显著高于静水组和低流速组(P<0.05);(3)中、高流速组血清中甘油三酯和总胆固醇显著...     

Relationships between pollutant discharge and water quality in the rivers from “better” to “worse” water quality

The long-term relationship between pollutant discharge and river water quality of biological/biochemical oxygen demand (BOD) in the Yamato-gawa River, Japan, has been found to be similar to perturbation or dynamic equilibrium change of alternative stable states in the ecosystem fields with a linear relationship as a usual condition. Similar relationships have been observed in short-terms for single precipitation events both in the Richmond River, Australia, and in a mountainous watershed in Japan. On the contrary, nutrient concentrations have not changed in a long-term besides nutrient discharge increases in the Richmond River. In this paper, long-term chronological relationships of annual pollutant discharges and water quality in the Brisbane River, Australia, were investigated based on pollutant discharge estimations and water quality monitoring data as a case study in the “better” water quality river. Effects of precipitation variation were taken into account in the pollutant discharge estimations by introducing precipitation variance coefficient (PVC). Total nitrogen (TN) and total phosphorus (TP) concentrations in the river were found to be improved with fluctuations in the lower Brisbane River Catchment during 1975–2012. Long-term dynamic equilibrium alterations of pollutant discharge and water quality were found for TN and TP at the first time in the Brisbane River. The investigated term was found to be divided into two phases, 1975–2001 and 2001–2012 for TN, and 1975–2007 and 2005–2012 for TP. Both pollutant discharges and pollutant concentrations in the river were smaller in Phase 2 than Phase 1. Based on the relationships in the three rivers, a hypothesis on pollutant discharge and water quality from “better” to “worse” river water environment was presented that rapid and/or excess pollutant discharge increases cause perturbation or dynamic equilibrium alterations with the basal relationships as the linear relationships.     

Metal biosorption in lignocellulosic biofuel biorefinery effluent: an initial step towards sustainability of water resources

Biosorption of metals by microorganisms is a promising technology to remove accumulated non-process elements in highly recycled biorefinery process water. Removal of these elements would enable greater water reuse and reduce the environmental impact of effluent discharge. A model lignocellulosic ethanol biorefinery wastewater was created based on pulp mill effluent. This generated a wastewater with an environmentally realistic high loading of dissolved natural organic matter (900?mg/l), a potentially important factor influencing metal biosorption. Analysis of feedstock and pulp mill effluent indicated that Mn and Zn are likely to be problematic in highly recycled lignocellulosic ethanol biorefinery process water. Therefore, the growth of several bacteria and fungi from existing collections, and some isolated from pulp mill effluent were tested in the model wastewater spiked with Mn and Zn (0.2?mM). Wastewater isolates grew the best in the wastewater. Metal uptake varied by species and was much greater for Zn than Mn. A bacterium, Novosphingobium nitrogenifigens Y88(T), removed the most metal per unit biomass, 35 and 17?mg?Mn/g. No other organism tested decreased the Mn concentration. A yeast, Candida tropicalis, produced the most biomass and removed the most total metal (38?% of Zn), while uptake per unit biomass was 24?mg?Zn/g. These results indicate that microorganisms can remove significant amounts of metals in wastewater with high concentrations of dissolved natural organic matter. Metal sorption by autochthonous microorganisms in an anaerobic bioreactor may be able to extend water reuse and therefore lower the water consumption of future biorefineries.     

Geochemical and Visual Indicators of Hydrothermal Fluid Flow through a Sediment-Hosted Volcanic Ridge in the Central Bransfield Basin (Antarctica)

In the austral summer of 2011 we undertook an investigation of three volcanic highs in the Central Bransfield Basin, Antarctica, in search of hydrothermal activity and associated fauna to assess changes since previous surveys and to evaluate the extent of hydrothermalism in this basin. At Hook Ridge, a submarine volcanic edifice at the eastern end of the basin, anomalies in water column redox potential (E_h) were detected close to the seafloor, unaccompanied by temperature or turbidity anomalies, indicating low-temperature hydrothermal discharge. Seepage was manifested as shimmering water emanating from the sediment and from mineralised structures on the seafloor; recognisable vent endemic fauna were not observed. Pore fluids extracted from Hook Ridge sediment were depleted in chloride, sulfate and magnesium by up to 8% relative to seawater, enriched in lithium, boron and calcium, and had a distinct strontium isotope composition (⁸⁷Sr/⁸⁶Sr  = 0.708776 at core base) compared with modern seawater (⁸⁷Sr/⁸⁶Sr ≈0.70918), indicating advection of hydrothermal fluid through sediment at this site. Biogeochemical zonation of redox active species implies significant moderation of the hydrothermal fluid with in situ diagenetic processes. At Middle Sister, the central ridge of the Three Sisters complex located about 100 km southwest of Hook Ridge, small water column E_h anomalies were detected but visual observations of the seafloor and pore fluid profiles provided no evidence of active hydrothermal circulation. At The Axe, located about 50 km southwest of Three Sisters, no water column anomalies in E_h, temperature or turbidity were detected. These observations demonstrate that the temperature anomalies observed in previous surveys are episodic features, and suggest that hydrothermal circulation in the Bransfield Strait is ephemeral in nature and therefore may not support vent biota.