The flocculation of bacteria using cationic synthetic flocculants and chitosan

Summary Bacteria are flocculated with high molecular weight cationic synthetic flocculants and chitosan. High charge density polymers are the most effective of the synthetic flocculants. Only chitosan is effective in flocculating the E. coli and B. subtilis cultures in complex broth. The difference in effectiveness between the synthetic flocculants and chitosan for flocculating E. coli, B. subtilis and Z. mobilis may be attributed to hydrogen bonding between the polysaccharide flocculant and cell surface polymers in addition to electrostatic interactions, and, in complex media, complexation of synthetic polymers with anionic polyelectrolytes.     

Detection of oxytocin,atrial natriuretic peptide,and brain natriuretic peptide using novel imprinted polymers produced with amphiphilic monomers

The cystine‐bridged cyclic peptide hormones (CBCPHs) represent signature structural feature as well as unique biological activity. In this study, three CBCPHs have been identified and characterized, namely, oxytocin, atrial natriuretic peptides (ANPs), and brain natriuretic peptides (BNPs). Because research has shown that ANPs and BNPs are powerful diagnostic biomarkers for heart disease, a highly laudable endeavor would be to develop a novel sensor for detecting ANP or BNP levels. Therefore, an amphiphilic monomer Acr‐His‐NHNH‐Fmoc was synthesized to form molecularly imprinted polymers (MIPs) for targeted CBCPH detection. First, oxytocin, a cardiovascular hormone and a CBCPH, was used as a template to fabricate MIPs on quartz crystal microbalance (QCM) chips. On the other hand, fabricated selected ANP segment or BNP segment as an epitope is able to construct epitope‐mediated MIPs (EMIPs) for ANP or BNP. The developed oxytocin or ANP sensor reached a detection limitation of 0.1nM with the dissociation constants being 30pM for oxytocin and 20pM for ANP. Moreover, BNP sensor achieved a detection limitation of 2.89pM with an even lower K_d value as 2pM. Compared with the performance of EMIPs, the imprinted films showed high affinity and selectivity in special binding to CBCPHs. The developed MIPs‐QCM biosensors thus provide an improved sensing platform using an amphiphilic monomer and may be useful for applications toward cyclotides, cystine knot motifs, or insulin‐like peptides.     

Calcium-Mediated Responses of Maize to Oxygen Deprivation

Oxygen limitation dramatically alters the patterns of gene expression as well as development of plants. Complete removal of O₂ leads to an immediate cessation of protein synthesis followed by a selective synthesis of about twenty anaerobic proteins in maize (Zea mays L.) seedlings. Among these are enzymes involved in glycolysis and related processes. However, inducible genes that have different functions were also found; they may function in other, perhaps more long-term, processes of adaptations to flooding, such as aerenchyma formation and root-tip death. Our recent research has addressed two questions: how these gene expression changes are initiated and how do these responses culminate in the overall adaptation of plants to flooding-stress. The results obtained indicate that an early rise in cytosolic Ca²⁺ as well as a quick establishment of ionic homeostasis may be essential for the induction of adaptive changes at the cellular as well as organismal level.     

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions

Inorganic phosphate (P_i) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to P_i limitation have been reported in E. coli. Given the essential role of P_i, adaptation to P_i limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting P_i conditions for 75 days. Not surprisingly, adapted cells showed better survival under P_i limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with P_i (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term P_i limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U‐¹³C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to P_i limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749–758, 2017     

Inducing flocculation of non-floc-forming <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

The present article reviews several approaches for inducing flocculation of Escherichia coli cells. The common industrially used bacterium E. coli does not naturally have floc-forming ability. However, there are several approaches to induce flocculation of E. coli cells. One is induction by flocculants—polyvalent inorganic salts, synthetic polymeric flocculants, or bio-based polymeric materials, including polysaccharide derivatives. Another method is the induction of spontaneous flocculation by changing the phenotypes of E. coli cells; several studies have shown that physical treatment or gene modification can endow E. coli cells with floc-forming ability. Coculturing E. coli with other microbes is another approach to induce E. coli flocculation. These approaches have particular advantages and disadvantages, and remain open to clarification of the flocculation mechanisms and improvement of the induction processes. In this review, several approaches to the induction of E. coli flocculation are summarized and discussed. This review will be a useful guide for the future development of methods for the flocculation of non-floc-forming microorganisms.     

Microbial community responses reduce soil carbon loss in Tibetan alpine grasslands under short‐term warming

Changes in labile carbon (LC) pools and microbial communities are the primary factors controlling soil heterotrophic respiration (R_h) in warming experiments. Warming is expected to initially increase R_h but studies show this increase may not be continuous or sustained. Specifically, LC and soil microbiome have been shown to contribute to the effect of extended warming on R_h. However, their relative contribution is unclear and this gap in knowledge causes considerable uncertainty in the prediction of carbon cycle feedbacks to climate change. In this study, we used a two‐step incubation approach to reveal the relative contribution of LC limitation and soil microbial community responses in attenuating the effect that extended warming has on R_h. Soil samples from three Tibetan ecosystems—an alpine meadow (AM), alpine steppe (AS), and desert steppe (DS)—were exposed to a temperature gradient of 5–25°C. After an initial incubation period, soils were processed in one of two methods: (a) soils were sterilized then inoculated with parent soil microbes to assess the LC limitation effects, while controlling for microbial community responses; or (b) soil microbes from the incubations were used to inoculate sterilized parent soils to assess the microbial community effects, while controlling for LC limitation. We found both LC limitation and microbial community responses led to significant declines in R_h by 37% and 30%, respectively, but their relative contributions were ecosystem specific. LC limitation alone caused a greater R_h decrease for DS soils than AMs or ASs. Our study demonstrates that soil carbon loss due to R_h in Tibetan alpine soils—especially in copiotrophic soils—will be weakened by microbial community responses under short‐term warming.     

The growth of sickle hemoglobin polymers

The measurement of polymer growth is an essential element in characterization of assembly. We have developed a precise method of measuring the growth of sickle hemoglobin polymers by observing the time required for polymers to traverse a photolytically produced channel between a region in which polymers are created and a detection region. The presence of the polymer is functionally detected by observing its ability to create new polymers through the well-established process of heterogeneous nucleation. Using this method, we have determined the rate constants for monomer addition to and release from polymer ends, as well as their temperature dependences. At 25°C we find k₊ = 84 ± 2 mM⁻¹ s⁻¹ and k₋ = 790 ± 80 molecules/s from each end. These numbers are in accord with differential interference contrast measurements, and their ratio gives a solubility measured on individual fibers. The single-fiber solubility agrees with that measured in sedimentation experiments. The concentration dependence of the monomer addition rate is consistent with monomer addition, but not oligomer addition, to growing polymers. The concentration dependence suggests the presence of an activation enthalpy barrier, and the rate of monomer addition is not diffusion-limited. Analysis of the temperature dependence of the monomer addition rate reveals an apparent activation energy of 9.1 ± 0.6 kcal/mol.     

Factors affecting the flocculation of bacteria by chemical additives

Pure bacterial cultures can be flocculated by a variety of chemical flocculants. Flocculation of bacteria will assist in their recovery, especially where the cells themselves are of interest, as in microbial protein production. Studies with several genera of bacteria indicate that the mechanism of flocculation is highly complex. Such interacting variables as temperature, ionic environment, physiological age, flocculant, bacterial genus, and surface shear have been observed. Jar test experiments with washed cells indicate that many of the variables are related to the release by the cell of proteins, nucleic acids, or polysaccharides. When released, these polymers may increase the required dosage of flocculant for recovery as in the case of E. coli, or the dosage may decrease as it does for Lactobacillus.     

Heterogeneity of leaf CO2 assimilation during photosynthetic induction

Spatial mid temporal variations in the distribution of photosynthesis over the leaf area were investigated during induction upon illumination of Rosa rubiginosa L. leaves. Gas exchange and maps of relative photosynthetie electron transport activity computed from chlorophyll fluorescence images were simultaneously monitored. In air, after 15 h of dark adaptation, linear electron transport was heterogeneously distributed over the leaf area during the induction. This patchy induction was explained by asynchronous metabolism activation for the first 10 min of illumination, concomitant asynchronous limitation by intrinsic metabolism and stomatal apertures (10–30 min) and finally by only stomatal limitation beyond 30 min. A brief transition to non-photorespiratory conditions after 20 min of illumination under subsaturating irradiance revealed a marked heterogeneity of CO₂ assimilation, presumably as a result of heterogeneous stomatal apertures. The frequency distribution of CO₂ assimilation was unimodal. During the induction, heterogeneity gradually decreased and photosynthesis was uniform at steady-state. After 10 min of dark adaptation, heterogeneity of linear electron transport activity occurred during the first 15 min of a second induction and mainly resulted from metabolic limitation.     

15N-NMR spectroscopy. IV. Comparison of poly(L-lysine) and isopoly(L-lysine)

The natural abundance ¹⁵N-nmr spectroscopy has been used to characterize the isomeric polymers (L -Lys)_n and iso (L -Lys)_n in aqueous solution. Although the peptide nitrogens of the two polymers have nearly equivalent shifts at pH < 10, the amino nitrogens differ by 5–6 ppm at pH < 7 and provide an easy means of identification. Furthermore, the polymers are distinguishable by the pK_a of the amino group and the basicity of the peptide nitrogen. At pH 10.3 and 25°C, (Lys)_n exhibits line broadening and an upfield chemical shift of the peptide nitrogen, indicative of the coil → helix transition. The formation of 100% helix may produce a shift as large as 5 ppm, which probably makes ¹⁵N-nmr spectroscopy more suitable for studies of this transition.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Phosphorus mining for ecological restoration on former agricultural land

To restore species‐rich terrestrial ecosystems on ex‐agricultural land, establishing nutrient limitation for dominant plant growth is essential because in nutrient‐rich soils, fast‐growing species often exclude target species. However, N‐limitation is easier to achieve than P‐limitation (because of a difference in biogeochemical behavior), biodiversity is generally highest under P‐limitation. Commonly used restoration methods to achieve low soil P‐concentrations are either very expensive or take a very long time. A promising restoration technique is P‐mining, an adjusted agricultural technique that aims at depleting soil‐P. High biomass production and hence high P‐removal with biomass are obtained by fertilizing with nutrients other than P. A pot experiment was set up to study P‐mining with Lolium perenne L. on sandy soils with varying P‐concentrations: from an intensively used agricultural soil to a soil near the soil P‐target for species‐rich Nardus grassland. All pots received N‐ and K‐fertilization. The effects of biostimulants on P‐uptake were also assessed by the addition of arbuscular mycorrhiza (Glomus spp.), humic substances or phosphate‐solubilizing bacteria (Bacillus sp. and Pseudomonas spp.). In our P‐rich soil (111 µg P_Olsen/g), P‐removal rate was high but bioavailable soil‐P did not decrease. At lower soil P‐concentrations (64 and 36 µg P_Olsen/g), bioavailable soil‐P had decreased but the P‐removal rate had by then dropped 60% despite N‐ and K‐fertilization and despite that the target (<10 µg P_Olsen/g) was still far away. None of the biostimulants altered this trajectory. Therefore, restoration will still take decades when starting with ex‐agricultural soils unless P‐fertilization history was much lower than average.     

H+ transport and coupling by the F0 sector of the ATP synthase: Insights into the molecular mechanism of function

The F₀ sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F₁ sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F₀ sector is reviewed here. TheE. coli F₀ is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F₀ is reviewed. The structure of purified subunitc in chloroform-methanol-H₂O resembles that in native F₀, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed.     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Variable Substitution Rates of the 18 Domain Sequences in Artemia Hemoglobin

The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by a gene representing nine successive globin domains which have different sequences and are presumed to have been copied originally from a single-domain gene. Two different polymers exist as the result of a complete duplication of the nine-domain gene, allowing the formation of either homodimers or the heterodimer. The total population size of 18 domains comprising nine corresponding pairs, coupled with the probability that they reflect several hundred million years of evolution in the same lineage, provides a unique model in which the process of gene multiplication can be analyzed. The outcome has important implications for the reliability of local molecular clocks. The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding individual domains are compared between polymers, amino acid substitution fluctuates by a factor of 2.7-fold from lowest to highest. This variation is not obvious at the DNA level: Domain pair identity values fluctuate by 1.3-fold. Identity values are, however, uncorrected for multiple substitutions, and both silent and nonsilent changes are pooled. Therefore, to determine the variability in relative substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96–99) to determine estimates of nonsynonymous (K _A ) and synonymous (K _S ) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (K _S of 56.9%) far exceeded nonsilent substitutions (K _A of 6.7%); however, for corresponding domain pairs, K _A fluctuates by 2.3-fold and K _S by 1.7-fold. The large discrepancies reflected in the expressed protein have accrued within a single lineage and the implication is that divergence dates of different genera based on amino acid sequences, even with well-studied proteins of reasonable size, can be wrong by a factor well in excess of 2. Received: 4 June 1997 / Accepted: 17 December 1997     

Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: C/N ratio and light intensity

We investigated the effect of carbon/nitrogen (C/N) ratio on astaxanthin synthesis in Haematococcus pluvialis during photoautotrophic induction by continuous input of both CO₂–air mixture and intense light. When H. pluvialis was induced by constant irradiance induction at 200 μmol photon m⁻² s⁻¹, there was a positive correlation with astaxanthin content and C/N ratio, which was similar to the case for heterotrophic induction. Lower C/N ratios did not retard Haematococcus encystment, but did increase culture biomass, resulting in a decrease in astaxanthin production because of light limitation. However, induction using variable irradiance showed that reduction of astaxanthin production at low C/N ratios was successfully overcome by simply increasing the light intensity from 200 to 300 μmol photon m⁻² s⁻¹ to overcome the light limitation. This resulted in a greatly enhanced astaxanthin synthesis in proportion to cell density in cultures with low C/N ratios. Our results indicate that light intensity is more critical than C/N ratio in astaxanthin production by H. pluvialis during photoautotrophic induction.     

Contribution of Anionic vs. Neutral Polymers to the Formation of Green Rust 1 from γ-FeOOH Bioreduction

To assess the effect of polymeric substances on the biomineralization and stabilization of green rust (GR), the effect of two organic polymers on the transformation of lepidocrocite (γ-FeOOH) to GR vs. magnetite in presence of Shewanella putrefaciens was investigated. These two polymers, generally used as flocculants, are polyacrylic acid (PAA), which bears negatively charged carboxylic groups at neutral pH and is expected to react with cationic hydrolyzed iron species, and polyacrylamide (PAM), which is a neutral polymer that may develop hydrogen bonds with iron nanocolloids. The bioreduction of lepidocrocite by S. putrefaciens was performed under conditions known to yield either magnetite or GR. Each operational condition of interest was investigated with various polymer concentrations from 0.6 to 60 mg g^?1 Fe (10 to 1000 mg l^?1). The final product was characterized using X-ray diffraction and electronic microscopy. The results showed that the formation of GR is favored, with respect to magnetite, to a lesser extent with PAM than with PAA. These results indicated that polymers influence the chemical stability of GR and/or guide the route of biomineralization. Polymer properties, in addition to silica and phosphate concentrations, are then critical parameters that control the secondary iron mineral biomineralization from iron-reducing bacteria.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

During photosynthetic induction,biochemical and stomatal limitations differ between Brassica crops

Interventions to increase crop radiation use efficiency rely on understanding of how biochemical and stomatal limitations affect photosynthesis. When leaves transition from shade to high light, slow increases in maximum Rubisco carboxylation rate and stomatal conductance limit net CO₂ assimilation for several minutes. However, as stomata open intercellular [CO₂] increases, so electron transport rate could also become limiting. Photosynthetic limitations were evaluated in three important Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. Measurements of induction after a period of shade showed that net CO₂ assimilation by B. rapa and B. napus saturated by 10 min. A new method of analyzing limitations to induction by varying intercellular [CO₂] showed this was due to co-limitation by Rubisco and electron transport. By contrast, in B. oleracea persistent Rubisco limitation meant that CO₂ assimilation was still recovering 15 min after induction. Correspondingly, B. oleracea had the lowest Rubisco total activity. The methodology developed, and its application here, shows a means to identify the basis of variation in photosynthetic efficiency in fluctuating light, which could be exploited in breeding and bioengineering to improve crop productivity.     

Effect of iron and other nutrient limitations on the pattern of outer membrane proteins in the cyanobacterium Synechococcus PCC7942

When cells of Synechococcus PCC7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of M _r 92000, 48000–50000 and 35000 appeared. Specific iron-limited outer membrane proteins (IRMPs) of M _r 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC6308. Under magnesium limitation polypeptides of M _r 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of M _r 32000 and one of over 100000 being induced, whereas calcium limitation had no apparent affect.Abbreviations EDDA ethylenediaminedihydroxyphenyl acetic acid - IRMP iron-regulated outer membrane protein - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride