Euglena sp as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH4-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (mu) of 0.28 day(-1) and biomass productivities of 132 mg L-1 day(-1). The algae cultured within a short period of 8 days resulted in the 98 % removal of NH4-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g L-1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.

Bioremediation and lipid synthesis through mixotrophic algal consortia in municipal wastewater

Algae grown in outdoor reactors (volume: 10 L and depth: 20 cm) were fed directly with filtered and sterilised municipal wastewater. The nutrient removal efficiencies were 86%, 90%, 89%, 70% and 76% for TOC, TN, NH4-N, TP and OP, respectively, and lipid content varied from 18% to 28.5% of dry algal biomass. Biomass productivity of similar to 122 mg/l/d (surface productivity 24.4 g/m(2)/d) and lipid productivity of similar to 32 mg/l/d were recorded. Gas chromatography and mass spectrometry (GC-MS) analyses of the fatty acid methyl esters (FAME) showed a higher content of desirable fatty acids (bearing biofuel properties) with major contributions from saturates such as palmitic acid C16:0; similar to 40%] and stearic acid C18:0; similar to 34%], followed by unsaturates such as oleic acid C18:1(9); similar to 10%] and linoleic acid C18:2(9,12); similar to 5%]. The decomposition of algal biomass and reactor residues with an exothermic heat content of 123.4 J/g provides the scope for further energy derivation. (C) 2014 Elsevier Ltd. All rights reserved.

Water treatment potential in relation to phytoplankton dynamics in tropical sewage-fed urban water bodies

Urban water bodies frequently receive untreated sewage and water levels in such water bodies are maintained by daily inputs of sewage. They function as “variable-zone” anaerobic-aerobic lagoons suffering several macrophyte, biotic and abiotic stresses. We have studied two such lakes in Bangalore (Bellandur-360 ha and Varthur-220 ha) to understand whether such an occurrence could be made beneficial (maintaining water levels as well as treatment). Such hypertrophic water body receives sewage at 180-250mg/L and is discharged at 25-80mg/L COD/BOD in different seasons. In an earlier study we reported macrophyte altering the purification function of the water body. In this paper we studied the impact of phytoplankton dynamics and macrophyte cover on the functions such as organic load removal. Algal community analysis, algal biomass, macrophyte cover, water quality, nutrient status was studied seasonally during 2009-2010. Oxygen deficiency and sometimes anoxia, recorded from surface samples resulted in high quantities of NH4+-N (30-40mg/L) and phosphate (0.5-4mg/L)-characteristics of anoxic hypertrophic urban lakes. The productiveness favoured high phytoplanktonic community characterized by small cells (<10μm; Chlorella sp. - highest numbers). The lake could be clearly demarcated into an initial anaerobic zone (40% area), a facultative zone (20%) and an aerobic zone (40%) based on redox values and GIS/bathymetry. During summer the lake is covered by floating macrophytes converting the lake into an anoxic/anaerobic water pool subduing the water purification function as well as aesthetics. When macrophytes are controlled such sewage fed water bodies can be used for treating urban wastewater while also maintaining water sustainability in these semi-arid ecosystems. This paper reports the community dynamics of phytoplankton, their function and competition with macrophytes.

Role of Protozoa in the Aerobic Purification of Sewage

DURING recent years, there has been increasing amount of evidence regarding the importance of protozoa in the aerobic purification of sewage1–4 and, more recently, some quantitative observations on protozoa in determining the condition of sludge and quality of effluent have been recorded5–10. The evidence so far obtained has, however, been only indirect, chiefly owing to the difficulty in separating the protozoa from the associated bacteria. This has now been achieved and the object of this note is to show that the isolated protozoa can bring about practically all the changes associated with the purification. The part played by the bacteria is almost negligible.

An enzymic approach to the study of sewage and sludges

The purpose of the work described here has been (a) to obtain some evidence on catalase (an oxidative enzyme) and protease, urease and phosphatase (hydrolytic enzymes) in sewage, activated sludge and septic tank sludge, and (b) to use this evidence, as a new approach, to find out the relationship between the main groups of the micro-organisms (bacteria and protozoa) and their relative influence on the purification process. To make a rapid assessment of the enzyme activities in these systems in the course of three weeks, as an experimental measure, rat tissues were added, which might serve as an additional or a ‘shock’ load of organic matter to follow broadly the development of bacteria and protozoa and the changes in the enzyme activities in the different systems. A control system with sewage alone was also run. The results showed that the initial decomposition of the fresh organic matter added to sewage and sludges was almost entirely due to bacterial activity and the later oxidative changes and removal of the suspended solids, including the bacteria, were largely due to the protozoa, such as Epistylis articulata. Analysis of the enzyme activities in the different materials showed, among other things, that the activated sludge, with its mized bacteria, protozoa and other organisms, as a whole, contained about twenty times more protease activity than an equivalent amount of the protozoan E. articulata, and that this protozoan contained five times more catalase activity than the activated sludge. The significance of these observations is discussed.

Purification of Antibodies Specific to a Dinucleotide Using the Hapten Bound to Deae Cellulose as an Affinity Column

The dinucleotide dpTpA held electrostatically on DEAE cellulose was used as an affinity column for the purification of dpTpA specific antibodies. Chromatography of the y-globulin fraction from dpTpA specific antisera on this column resulted in the retention of dpTpA specific antibodies which were later eluted along with the bound dpTpA using 1M NaC1. Dextran coated charcoal was the method of choice for the dissociation and removal of dpTpA bound to the antibodies. This method may extend itself to the purification of antibodies specific for other oligonucleotides.

Reuse of treated sewage in Delhi city: Microbial evaluation of STPs and reuse options

Microbiological quality of the treated wastewater is an important parameter for its reuse. The data oil the Fecal Coliform (FC) and Fecal Streptococcus (FS) at different stages of treatment in the Sewage Treatment Plants (STPs) in Delhi watershed is not available, therefore in the present study microbial profiling of STPs was carried out to assess the effluent quality for present and future reuse options. This Study further evaluates the water quality profiles at different stages of treatment for 16 STPs in Delhi city. These STPs are based on conventional Activated Sludge Process (ASP), extended aeration, physical, chemical and biological treatment (BIOFORE), Trickling Filter and Oxidation Pond. The primary effluent quality produced from most of the STPs was suitable for Soil Aquifer Treatment (SAT). Extended Hydraulic Retention Time (HRT) as a result Of low inflow to the STPS Was responsible for high turbidity, COD and BODs removal. Conventional ASP based STPs achieved 1.66 log FC and 1.06 log FS removal. STPs with extended aeration treatment process produced better quality effluent with maximum 4 log order reduction in FC and FS levels. ``Kondli'' and ``Nilothi'' STPs employing ASP, produced better quality secondary effluent as compared to other STPs based oil similar treatment process. Oxidation Pond based STPs showed better FC and FS removals, whereas good physiochemical quality was achieved during the first half of the treatment. Based upon physical, chemical and microbiological removal efficiencies, actual integrated efficiency (IEa) of each STP was determined to evaluate its Suitability for reuse for irrigation purposes. Except Mehrauli'' and ``Oxidation Pond'', effluents from all other STPs require tertiary treatment for further reuse. Possible reuse options, depending Upon the geographical location, proximity of facilities of potential users based oil the beneficial uses, and sub-soil types, etc. for the Delhi city have been investigated, which include artificial groundwater recharge, aquaculture, horticulture and industrial uses Such as floor washing, boiler feed, and cooling towers, etc. (C) 2009 Elsevier B.V. All rights reserved.

Dynamic simulation of activated sludge based wastewater treatment processes: Case studies with Titagarh Sewage Treatment Plant, India

STOAT has been extensively used for the dynamic simulation of an activated sludge based wastewater treatment plant in the Titagarh Sewage Treatment Plant, near Kolkata, India. Some alternative schemes were suggested. Different schemes were compared for the removal of Total Suspended Solids (TSS), b-COD, ammonia, nitrates etc. A combination of IAWQ#1 module with the Takacs module gave best results for the existing scenarios of the Titagarh Sewage Treatment Plant. The modified Bardenpho process was found most effective for reducing the mean b-COD level to as low as 31.4 mg/l, while the mean TSS level was as high as 100.98 mg/l as compared to the mean levels of TSS (92 62 mg/l) and b-COD (92.0 mg/l) in the existing plant. Scheme 2 gave a better scenario for the mean TSS level bringing it down to a mean value of 0.4 mg/l, but a higher mean value for the b-COD level at 54.89 mg/l. The Scheme Final could reduce the mean TSS level to 2.9 mg/l and the mean b-COD level to as low as 38.8 mg/l. The Final Scheme looks to be a technically viable scheme with respect to the overall effluent quality for the plant. (C) 2009 Elsevier B.V. All rights reserved.

A study of the purification and properties of tryptophan synthetase of Bengal gram (Cicer arietinum)

Active preparations of tryptophan synthetase were obtained from Bengal gram (Cicer arietinum) by the following procedure: (1) precipitation of inactive materials by manganous sulfate, (2) Adsorption of impurities on Alumina Cγ, (3) Adsorption of tryptophan synthetase on tricalcium phosphate gel, removal of inert protein from the gel by treatment with phosphate buffer (pH 7.2), and selective elution of the enzyme by 0.15 M phosphate buffer pH 7.2 containing 10% ammonium sulfate and 10−3 M serine. A 220-fold purification of the enzyme with 44% recovery of the activity was achieved. The pH optimum, effect of temperature, and substrate concentration and other properties of the purified enzyme have been studied in detail. Only the Image -isomer of serine takes part in the reaction. The Km values for indole, Image -serine, and Image -serine were calculated to be 0.66, 4.1, and 8.6 × 10−4 M, respectively. A kinetic study of the inhibition of tryptophan synthetase by indole-propionic acid has shown that it is of a competitive type. It has been demonstrated for the first time that 4-nitro-salicylaldehyde can replace pyridoxal phosphate as a coenzyme for the tryptophan synthetase reaction.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

CDNA Cloning, Overexpression in Escherichia coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The K-m values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.

Assessment of treatment capabilities of Varthur Lake, Bangalore, India

Manmade waterbodies have traditionally been used for domestic and irrigation purposes. Unplanned urbanization and ad-hoc approaches have led to these waterbodies receiving untreated sewage. This enriches and eutrophies the waterbody. A physicochemical and biological analysis of sewage-fed Varthur Lake in Bangalore was carried out and its treatment capabilities in terms of BOD removal, nutrient assimilation and self-remediation were assessed. Anaerobic conditions (0 mg/L) prevail at the inlet which improves towards the outlets due to algal aeration. This removed > 50% BOD in the monsoon season but was inhibited by floating macrophytes in all other seasons. Alkalinity, TDS, conductivity and hardness values were higher when compared to earlier studies. This study shows the lake behaves as an anaerobic~aerobic lagoon with a residence time of 4.8 d treating the wastewater to a considerable extent. Further research is required to optimise the system performance.

Treatment efficacy of algae-based sewage treatment plants

Lagoons have been traditionally used in India for decentralized treatment of domestic sewage. These are cost effective as they depend mainly on natural processes without any external energy inputs. This study focuses on the treatment efficiency of algae-based sewage treatment plant (STP) of 67.65 million liters per day (MLD) capacity considering the characteristics of domestic wastewater (sewage) and functioning of the treatment plant, while attempting to understand the role of algae in the treatment. STP performance was assessed by diurnal as well as periodic investigations of key water quality parameters and algal biota. STP with a residence time of 14.3 days perform moderately, which is evident from the removal of total chemical oxygen demand (COD) (60 %), filterable COD (50 %), total biochemical oxygen demand (BOD) (82 %), and filterable BOD (70 %) as sewage travels from the inlet to the outlet. Furthermore, nitrogen content showed sharp variations with total Kjeldahl nitrogen (TKN) removal of 36 %; ammonium N (NH4-N) removal efficiency of 18 %, nitrate (NO3-N) removal efficiency of 22 %, and nitrite (NO2-N) removal efficiency of 57.8 %. The predominant algae are euglenoides (in facultative lagoons) and chlorophycean members (maturation ponds). The drastic decrease of particulates and suspended matter highlights heterotrophy of euglenoides in removing particulates.

Exploring sand and bentonite - enhanced sand as filter media for nitrate removal

Increasing nitrate concentrations in ground water is deleterious to human health as ingestion of such water can cause methemoglobinemia in infants and even cancer in adults (desirable limit for nitrate as NO3 - 45 mg/L, IS code 10500-1991). Excess nitrate concentrations in ground water is contributed by reason being disposal of sewage and excessive use of fertilizers. Though numerous technologies such as reverse osmosis, ion exchange, electro-dialysis, permeable reactive barriers using zerovalent iron etc exists, nitrate removal continues to be one of challenging issue as nitrate ion is highly mobile within the soil strata. The tapping the denitrification potential of soil denitrifiers which are inherently available in the soil matrix is the most sustainable approach to mitigate accumulation of nitrate in ground water. The insitu denitrification of sand and bentonite enhanced sand (bentonite content = 5%) in presence of easily assimilable organic carbon such as ethanol was studied. Batch studies showed that nitrate reduction by sand follows first order kinetics with a rate constant 5.3x10(-2) hr(-1) and rate constant 4.3 x 10(-2) hr(-1) was obtained for bentonite-enhanced sand (BS) at 25 degrees C. Filter columns (height = 5 cm and diameter = 8.2 cm) were constructed using sand and bentonite-enhanced sand as filter media. The filtration rate through both the filter columns was maintained at average value of 2.60 cm/h. The nitrate removal rates through both the filter media was assessed for solution containing 22.6 mg NO3-N/L concentrations while keeping C/N mass ratio as 3. For sand filter column, the nitrate removal efficiency reached the average value of 97.6% after passing 50 pore volumes of the nitrate solution. For bentonite-enhanced sand filter column, the average nitrate removal efficiency was 83.5%. The time required for effective operation for sand filter bed was 100 hours, while bentonite-enhanced sand filter bed did not require any maturation period as that of sand filter bed for effective performance because the presence of micropores in bentonite increases the hydraulic retention time of the solution inside the filter bed.

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.