Evaluation of biologically mediated changes in oil sands naphthenic acid composition by Chlamydomonas reinhardtii using negative‐ion electrospray orbitrap mass spectrometry

Industrial activity associated with oil‐sands extraction in Canada's Athabasca region produces a variety of contaminants of concern, including naphthenic acid fraction components (NAFCs). NAFCs are a complex mixture of organic compounds that are poorly understood both in terms of their chemical composition and effects on the environment. NAFC toxicity in the unicellular green algae Chlamydomonas reinhardtii P.A.Dangeard was correlated with the presence of the algal cell wall. It was suggested that the toxicity of NAFCs in C. reinhardtii was due to surfactant effects. Surfactant‐cell wall interactions are specific and governed by the compound class and structure, and by the nature of the biological material. Here, we investigate the effects of wildtype (WT) C. reinhardtii and two cell‐wall mutants on specific classes of NAFCs when growing cultures were treated with a 100 mg · L^?1 solution of NAFCs. Changes in the NAFC composition in the media were examined using high resolution mass spectrometry over a period of 4 d. Algal mediated changes in the NAFCs were limited to specific classes of NAFCs. In particular, the removal of large, classical naphthenic acids, with a double bond equivalent of 8, was observed in WT C. reinhardtii cultures. The observed algal mediated changes in NAFC composition would have been masked by low resolution mass spectrometry and highlight the importance of this tool in examining bioremediation of complex mixtures of NAFCs.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

Action of antioxidant enzymes and cytochrome P-450 monooxygenases in the cabbage looper in response to plant phototoxins

Effects of two biosynthetically distinct plant phototoxins—xanthototoxin, a furanocoumarin, and harmine, a β-carboline alkaloid, which are known to produce toxic oxygen species—on the food utilization efficiencies and enzymatic detoxification systems of the polyphagous cabbage looper. Trichoplusia ni (Lepidoptera: Noctuidae), were studied. Newly molted fifth-instar larvae were allowed 36 h to ingest diets containing these two phototoxins at 0.15% wet weight in the presence of near ultraviolet (UVA). The growth and development of the larvae, as well as the corresponding activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) and the detoxification enzyme cytochrome P-450, were measured. Xanthotoxin reduced rates of relative growth and consumption and efficiencies of conversion of ingested and digested food to biomass. Harmine reduced rates of growth and consumption without affecting efficiencies of conversion. Specific activities of SOD, CAT, GPOX, and GR of whole-body homogenates in the absence of compounds were 0.88 units, 153μmol H₂O₂ decomposed·mg protein^?1·min—¹, 38.3 nmol NADPH oxidized·mg protein^?1·min^?1, and 0.56 nmol NADPH oxidized·mg protein^?1·min^?1, respectively. SOD activity was induced 2.9-fold and 3.8-fold by dietary xanthotoxin and harmine, respectively. CAT and GPOX activities were induced 1.2-fold by harmine only, and GR activity was not changed by either chemical. The P-450 activity toward xanthotoxin in the microsomal fraction of midguts was low (0.15 nmol xanthotoxin metabolized·mg protein^?1·min^?1) and was not induced by xanthotoxin ingestion. These studies indicate that P-450 and antioxidant enzyme systems may be independent but consequential, the induction of antioxidant enzymes by phototoxins occurring when low P-450 activity toward the phototoxin permits the accumulation of oxidative stress from unmetabolized phototoxin, which in turn induces antioxidant enzymes.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

Quantification of ventricular β2‐adrenoceptor density and ligand binding affinity in wild sockeye salmon Oncorhynchus nerka smolts using a novel modification to the tritiated ligand technique

A new, image‐based, tritiated ligand technique for measuring cardiac β₂‐adrenoceptor (β₂‐AR) binding characteristics was developed and validated with adult rainbow trout Oncorhynchus mykiss hearts so that the tissue limitation of traditional receptor binding techniques could be overcome and measurements could be made in hearts nearly 14‐times smaller than previously used. The myocardial cell‐surface (functional) β₂‐AR density of O. nerka smolts sampled at the headwaters of the Chilko River was 54·2 fmol mg protein^?1 and about half of that previously found in return migrating adults of the same population, but still more than twice that of adult hatchery O. mykiss (21·1 fmol mg protein^?1). This technique now opens the possibility of investigating cardiac receptor density in a much wider range of fish species and life stages.     

Inactivation of Bacillus cereus by Na‐chlorophyllin‐based photosensitization on the surface of packaging

Aims: This study was focused on the possibility to inactivate food‐borne pathogen Bacillus cereus by Na‐chlorophyllin (Na‐Chl)‐based photosensitization in vitro and after attachment to the surface of packaging material. Methods and Results: Bacillus cereus in vitro or attached to the packaging was incubated with Na‐Chl (7·5 × 10^?8 to 7·5 × 10^?5 mol l^?1) for 2–60 min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400 nm and an energy density of 20 mW cm^?2. The illumination time varied 0–5 min and subsequently the total energy dose was 0–6 J cm^?2. The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5 × 10^?7 mol l^?1 Na‐Chl and following illumination were inactivated by 7 log. The photoinactivation of B. cereus spores in vitro by 4 log required higher (7·5 × 10^?6 mol l^?1) Na‐Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5 log at 7·5 × 10^?5 mol l^?1 Na‐Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1 log, 100 ppm Na‐hypochlorite reduces the pathogens about 1·7 log and 200 ppm Na‐hypochlorite by 2·2 log. Meanwhile, Na‐Chl‐based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. Conclusions: Food‐borne pathogen B. cereus could be effectively inactivated (7 log) by Na‐Chl‐based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization‐based inactivation. Comparison of different surface decontamination treatments indicates that Na‐Chl‐based photosensitization is much more effective antibacterial tool than washing with water or 200 ppm Na‐hypochlorite. Significance and Impact of the Study: Our data support the idea that Na‐Chl‐based photosensitization has great potential for future application as an environment‐friendly, nonthermal surface decontamination technique.     

Generation of a phage‐display library of single‐domain camelid VHH antibodies directed against Chlamydomonas reinhardtii antigens,and characterization of VHHs binding cell‐surface antigens

Single‐domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain‐only variable V_H domain (V_HH) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and V_HH antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V_HH region contained at least 10⁶ individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for V_HH clones with specific recognition of cell‐surface epitopes. The lead candidate V_HH clones (designated B11 and H10) bound to C. reinhardtii with EC₅₀ values ≤0.5 nm . Treatment of cells with V_HH B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V_HH antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.     

THE SHORT‐TERM EFFECT OF IRRADIANCE ON THE PHOTOSYNTHETIC PROPERTIES OF ANTARCTIC FAST‐ICE MICROALGAL COMMUNITIES1

Although sea‐ice represents a harsh physicochemical environment with steep gradients in temperature, light, and salinity, diverse microbial communities are present within the ice matrix. We describe here the photosynthetic responses of sea‐ice microalgae to varying irradiances. Rapid light curves (RLCs) were generated using pulse amplitude fluorometry and used to derive photosynthetic yield (Φ_PSII), photosynthetic efficiency (α), and the irradiance (E_k) at which relative electron transport rate (rETR) saturates. Surface brine algae from near the surface and bottom‐ice algae were exposed to a range of irradiances from 7 to 262 μmol photons · m^?2 · s^?1. In surface brine algae, Φ_PSII and α remained constant at all irradiances, and rETR_max peaked at 151 μmol photons · m^?2 · s^?1, indicating these algae are well acclimated to the irradiances to which they are normally exposed. In contrast, Φ_PSII, α, and rETR_max in bottom‐ice algae reduced when exposed to irradiances >26 μmol photons · m^?2 · s^?1, indicating a high degree of shade acclimation. In addition, the previous light history had no significant effect on the photosynthetic capacity of bottom‐ice algae whether cells were gradually exposed to target irradiances over a 12 h period or were exposed immediately (light shocked). These findings indicate that bottom‐ice algae are photoinhibited in a dose‐dependent manner, while surface brine algae tolerate higher irradiances. Our study shows that sea‐ice algae are able to adjust to changes in irradiance rapidly, and this ability to acclimate may facilitate survival and subsequent long‐term acclimation to the postmelt light regime of the Southern Ocean.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

OPTIMIZATION OF IRON‐DEPENDENT CYANOBACTERIAL (SYNECHOCOCCUS,CYANOPHYCEAE) BIOREPORTERS TO MEASURE IRON BIOAVAILABILITY1

Complex chemistry and biological uptake pathways render iron bioavailability particularly difficult to assess in natural waters. Bioreporters are genetically modified organisms that are useful tools to directly sense the bioavailable fractions of solutes. In this study, three cyanobacterial bioreporters derived from Synechococcus PCC 7942 were examined for the purpose of optimizing the response to bioavailable Fe. Each bioreporter uses a Fe‐regulated promoter (isiAB, irpA and mapA), modulated by distinct mechanisms under Fe deficiency, fused to a bacterial luciferase (luxAB). In order to provide a better understanding of the way natural conditions may affect the ability of the bioreporter to sense iron bioavailability, the effect of relevant environmental parameters on the response to iron was assessed. Optimal conditions (and limits of applicability) for the use of these bioreporters on the field were determined to be: a 12 h (12–24 h) exposure time, temperature of 15°C (15°C–22°C), photon flux density of 100 μmol photons·m^?2·s^?1 (37–200 lmol photons·m^?2·s^?1), initial biomass of 0.6–0.8 lg chlorophyll a (chl a)·L^?1 (0.3–1.5 lg chl a·L^?1) or approximately 10⁵ bioreporter cells·mL^?1, high phosphate (10 lM), and low micronutrients (absent). The measured luminescence was optimal with an exogenous addition of 60 lM aqueous decanal substrate allowing a 5 min reaction time in the dark before analysis. This study provides important considerations relating to the optimization in the use of bioreporters under field conditions that can be used for method development of other algal and cyanobacterial bioreporters in aquatic systems.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Short‐term survival and movements of Atlantic sharpnose sharks captured by hook‐and‐line in the north‐east Gulf of Mexico

Ultrasonic telemetry was used to compare post‐release survival and movements of Atlantic sharpnose sharks Rhizoprionodon terraenovae in a coastal area of the north‐east Gulf of Mexico. Ten fish were caught with standardized hook‐and‐line gear during June to October 1999. Atlantic sharpnose sharks were continuously tracked after release for periods of 0·75 to 5·90 h and their positions recorded at a median interval of 9 min. Individual rate of movement was the mean of all distance and time measurements for each fish. Mean ± s.e . individual rate of movement was 0·45 ± 0·06 total lengths per second (L_T s^?1) and ranged from 0·28 to 0·92 L_T s^?1 over all fish. Movement patterns did not differ between jaw and internally hooked Atlantic sharpnose sharks. Individual rate of movement was inversely correlated with bottom water temperature at capture (r² = 0·52, P ≤ 0·05). No consistent direction in movement was detected for Atlantic sharpnose sharks after release, except that they avoided movement towards shallower areas. Capture‐release survival was high (90%), with only one fish not surviving, i.e. this particular fish stopped movement for a period of 10 min. Total rate of movement was total distance over total time (m min^?1) for each Atlantic sharpnose shark. Mean total rate of movement was significantly higher immediately after release at 21·5 m min^?1 over the first 1·5 h of tracking, then decreased to 11·2 m min^?1 over 1·5–6 h, and 7·7 m min^?1 over 3–6 h (P ≤ 0·002), which suggested initial post‐release stress but quick recovery from capture. Thus, high survival (90%) and quick recovery indicate that the practice of catch‐and‐release would be a viable method to reduce capture mortality for R. terraenovae.     

EFFECT OF LIGHT AND TEMPERATURE INTERACTIONS ON GROWTH OF CRYPTOMONAS EROSA (CRYPTOPHYCEAE)1

Cryptomonas erosa Skuja, a planktonic alga, was grown in batch culture at different combinations of light intensity and temperature, under nutrient saturation. Growth was maximal (1.2 divisions · day^?1) at 23.5 C and 0.043 ly · min^?1, declining sharply with temperature (0.025 divisions-day^?1 at 1 C). With decreasing temperature, the cells showed both light saturation and inhibition at much reduced light intensities. At the same time the compensation light intensity for growth declined towards a minimum of slightly above 0.4 × 10^?4 ly · min^?1 (~1 ft-c) at 1 C or <0.1 ly · day^?1 (PAR). Cell division was more adversely affected by low temperature than carbon uptake, and the resulting excess production of photosynthate was both stored and excreted. Extreme storage of carbohydrates resulted in cell volumes and carbon content ca. 22 and 30 × greater, respectively, than the maxima observed for cells incubated in the dark, whereas, at growth inhibitory light levels, as much as 57% of the total assimilated carbon was excreted. A marked increase in cell pigment was observed at the lowest light levels (<10^?3 ly · min^?1), at high temperature. The growth response of C. erosa in culture provides insight into the abundance and distribution of cryptomonads and other small algal flagellates in nature.     

PRODUCTION AND NUTRIENT REMOVAL BY PERIPHYTON GROWN UNDER DIFFERENT LOADING RATES OF ANAEROBICALLY DIGESTED FLUSHED DAIRY MANURE1

Growing algae to scrub nutrients from manure presents an alternative to the current practice of land application and provides utilizable algal biomass as an end product. The objective of this study was to assess algal growth, nutrient removal, and nitrification using higher light intensities and manure loading rates than in the previous experiments. Algal turfs, with periphyton mainly composed of green algal species, were grown under two light regimes (270 and 390 μmol photons·m^?2· s^?1) and anaerobically digested flushed dairy manure wastewater (ADFDMW) loading rates ranging from 0.8 to 3.7 g total N and 0.12 to 0.58 g total P·m^?2·d^?1. Filamentous cyanobacteria (Oscillatoria spp.) and diatoms (Navicula, Nitzschia, and Cyclotella sp.) partially replaced the filamentous green algae at relatively high ADFDMW loading rates and more prominently under low incident light. Mean algal production increased with loading rate and irradiance from 7.6±2.71 to 19.1±2.73 g dry weight· m^?2·d^?1. The N and P content of algal biomass generally increased with loading rate and ranged from 2.9%–7.3% and 0.5%–1.3% (by weight), respectively. Carbon content remained relatively constant at all loading rates (42%–47%). The maximum removal rates of N and P per unit algal biomass were 70 and 13 mg·g^?1 dry weight·m^?2·d^?1, respectively. Recovery of nutrients in harvested algal biomass accounted for about 31%–52% for N and 30%–59% for P. Recovery of P appeared to be uncoupled with N at higher loading rates, suggesting that algal potential for accumulation of P may have already been saturated. It appears that higher irradiance level enhancing algal growth was the overriding factor in controlling nitrification in the algal turf scrubber units.     

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Inulin is a linear carbohydrate polymer of fructose subunits (2‐60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo‐oligosaccharides, biofuel, and nutraceuticals. Cell‐free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo‐inulinase was investigated in this study. The eukaroyototic exo‐inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta‐gami B (DE3)]. The molecular weight of recombinant exo‐inulinase was estimated to be ～81 kDa. The values of K_m and V_max of the recombinant exo‐inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min^?1 mg^?1 protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min^?1 mg^?1 protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo‐inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo‐inulinase activity towards inulin was enhanced by Cu²⁺ and reduced by Fe²⁺, while its activity towards sucrose was enhanced by Co²⁺ and reduced by Zn²⁺. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629–637, 2016     

Synthesis and assembly of a full‐length human monoclonal antibody in algal chloroplasts

Monoclonal antibodies can be effective therapeutics against a variety of human diseases, but currently marketed antibody‐based drugs are very expensive compared to other therapeutic options. Here, we show that the eukaryotic green algae Chlamydomonas reinhardtii is capable of synthesizing and assembling a full‐length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts. This antibody, 83K7C, is derived from a human IgG1 directed against anthrax protective antigen 83 (PA83), and has been shown to block the effects of anthrax toxin in animal models. Here we show that 83K7C heavy and light chain proteins expressed in the chloroplast accumulate as soluble proteins that assemble into complexes containing two heavy and two light chain proteins. The algal‐expressed 83K7C binds PA83 in vitro with similar affinity to the mammalian‐expressed 83K7C antibody. In addition, a second human IgG1 and a mouse IgG1 were also expressed and shown to properly assemble in algal chloroplast. These results show that chloroplasts have the ability to fold and assemble full‐length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins. Biotechnol. Bioeng. 2009; 104: 663–673 © 2009 Wiley Periodicals, Inc.