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Applied Microbiology and Biotechnology (v.83, #6)

The metabolomics of carotenoids in engineered cell factory by Guan-Nan Liu; Yue-Hui Zhu; Jian-Guo Jiang (pp. 989-999).

Carotenoids such as β-carotene, lycopene, and antheraxanthin have plenty of scientific and commercial value. The comprehensive investigation of carotenoids drives people to improve and develop all kinds of analytical techniques to approach or even achieve “versatile” analysis. The metabolic engineering efforts in plants and algae have progressed rapidly, aiming to enable the use of plants and algae as “cell factories” for producing specific or novel carotenoids, such as β-carotene (provitamin A) in Gold rice, while the emerging technologies of metabolomics support it by providing comprehensive analysis of carotenoids biochemical characterizations. This review describes metabolomics as a high-throughput platform to study carotenoids, including the engineering methods in the plants or algae, the bioinformatics for metabolomics, and the metabolomics of carotenoids in engineered cell factory. Modern systems biology tools, together with the development of genomics and metabolomics databases, will dramatically facilitate the advancement of our knowledge in gene-to-metabolite networks in plants. Metabolomics accompanying genomics, transcriptomics, and proteomics as well as bioinformatics facilitate metabolic engineering efforts towards designing superior biocatalysts in cell factories. Ongoing advances in biological techniques coupled with crucial metabolic networks will further promote plants and algae as attractive platforms for the production of numerous high-value compounds such as carotenoids.

Keywords: Carotenoids; Bioinformatics; Target analysis; Metabolic profiling; Metabolic fingerprinting; Metabolomics; Metabolomic analysis techniques

Production of Chlorella biomass enriched by selenium and its use in animal nutrition: a review by Jiří Doucha; Karel Lívanský; Václav Kotrbáček; Vilém Zachleder (pp. 1001-1008).

Feedstuffs are routinely supplemented with various selenium sources, where organic forms of Se are more bio-available and less toxic than the inorganic forms (selenites, selenates). When the algae are exposed to environmental Se in the form of selenite, they are able as other microorganisms to incorporate the element to different levels, depending on the algae species. Technology of heterotrophic fed-batch cultivation of the microalga Chlorella enriched by organically bound Se was developed, where the cultivation proceeds in fermentors on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. High volumetric productivity and high cell concentrations (about 70–100 g Chlorella dry mass l⁻¹) can be attained if nutrients and oxygen are adequately supplied. Addition of a small quantity of a new selenoprotein source-spray-dried Se-Chlorella biomass to the diet of farm animals had better effects on specific physiological and physical parameters of animals than selenite salt and was comparable with Se yeast added to the diet. This review introduces the importance of selenium for humans and animals, methods of Se determination, heterotrophic production of selenium-enriched Chlorella biomass in a fed-batch culture regime on organic carbon, and use of the biomass in animal nutrition.

Keywords: Chlorella ; Heterotrophic production; Nutrition; Organic-bound selenium; Se determination

Synthesis and characterization of hydroquinone fructoside using Leuconostoc mesenteroides levansucrase by Jin Kang; Young-Min Kim; Nahyun Kim; Du-Woon Kim; Seung-Hee Nam; Doman Kim (pp. 1009-1016).

Hydroquinone (HQ) functions as a skin-whitening agent, but it has the potential to cause dermatitis. We synthesized a HQ fructoside (HQ-Fru) as a potential skin-whitening agent by reacting levansucrase from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a fructofuranose donor. The product was purified using 1-butanol partition and silica-gel column chromatography. The structure of the purified HQ-Fru was determined by ¹H and ¹³C nuclear magnetic resonance, and the molecular ion of the product was observed at m/z 295 (C12 H16 O7 Na)⁺. The HQ-Fru was identified as 4-hydroxyphenyl-β-d-fructofuranoside. The optimum condition for HQ-Fru synthesis was determined using a response surface method (RSM), and the final optimum condition was 350 mM HQ, 115 mM sucrose, and 0.70 U/ml levansucrase, and the final HQ-Fru produced was 1.09 g/l. HQ-Fru showed anti-oxidation activities and inhibition against tyrosinase. The median inhibition concentration (IC₅₀) of 1,1-diphenyl-2-picrylhydrazyl scavenging activity was 5.83 mM, showing higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). The K i value of HQ-Fru (1.53 mM) against tyrosinase was smaller than that of β-arbutin (K i = 2.8 mM), indicating that it was 1.8-times better as an inhibitor. The inhibition of lipid peroxidation by HQ-Fru was 105.3% that of HQ (100%) and 118.9 times higher than that of β-arbutin (0.89% of HQ).

Keywords: Leuconostoc mesenteroides ; Levansucrase; Hydroquinone; Acceptor reaction; Fructosylation

Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties by Tomotake Morita; Tokuma Fukuoka; Masaaki Konishi; Tomohiro Imura; Shuhei Yamamoto; Masaru Kitagawa; Atsushi Sogabe; Dai Kitamoto (pp. 1017-1025).

The development of a novel glycolipid biosurfactant was undertaken using the high-level producers of mannosylerythritol lipids (MELs) such as Pseudozyma parantarctica, Pseudozyma antarctica, and Pseudozyma rugulosa. Besides the conventional MELs (MEL-A, MEL-B, and MEL-C), these yeasts produced an unknown glycolipid when they were cultivated in a medium containing 4% (w/v) olive oil and 4% (w/w) mannitol as the carbon source. The unknown glycolipid extracted from the culture medium of P. parantarctica JCM 11752^T displayed the spot with lower mobility than that of known MELs on TLC and provided mainly two peaks identical to mannose and mannitol on high-performance liquid chromatography after acid hydrolysis. Based on structural analysis by ¹H and ¹³C nuclear magnetic resonance, the novel glycolipid was composed of mannose and mannitol as the hydrophilic sugar moiety and was identified as mannosylmannitol lipid (MML). Of the strains tested, P. parantarctica JCM 11752^T gave the best yield of MML (18.2 g/L), which comprised approximately 35% of all glycolipids produced. We further investigated the interfacial properties of the MML, considering the unique hydrophilic structure. The observed critical micelle concentration (CMC) and the surface tension at CMC of the MML were 2.6 × 10⁻⁶ M and 24.2 mN/m, respectively. In addition, on a water-penetration scan, the MML efficiently formed not only the lamella phase (Lα) but also the myelins at a wide range of concentrations, indicating its excellent self-assembling properties and high hydrophilicity. The present glycolipid should thus facilitate the application of biosurfactants as new functional materials.

Keywords: Mannosylmannitol lipid; Biosurfactant; Glycolipid; Pseudozyma

Chemically assisted microbial production of succinic acid by the yeast Yarrowia lipolytica grown on ethanol by Svetlana V. Kamzolova; Alsu I. Yusupova; Natalia G. Vinokurova; Nadezhda I. Fedotcheva; Maria N. Kondrashova; Tatiana V. Finogenova; Igor G. Morgunov (pp. 1027-1034).

A new two-step process of production of succinic acid (SA) has been developed, which includes the microbial synthesis of alpha-ketoglutaric acid by the yeast Yarrowia lipolytica (step 1) and subsequent oxidation of the acid by hydrogen peroxide to SA (step 2). The maximum concentration of SA and its yield were found to be 63.4 g l⁻¹ and 58% of the ethanol consumed, respectively. The purity of the SA isolated from the culture liquid filtrate reached 100%. The yield of SA was as high as 82% of its amount in the culture liquid filtrate. The quality of the SA produced by the invented method meets the biochemical grade definitions, as is evident from the respiratory and other relevant parameters of rat liver mitochondria upon the oxidation of this SA.

Keywords: Microbial production; Succinic acid; α-Ketoglutaric acid; Yarrowia lipolytica ; Ethanol

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from Clostridium beijerinckii by Dae Haeng Cho; Yun Jie Lee; Youngsoon Um; Byoung-In Sang; Yong Hwan Kim (pp. 1035-1043).

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.

Keywords: Lignocellulosic hydrolysates; Phenolic compound; Peroxidase; Butanol; Clostridium beijerinckii ; Inhibitor

A periplasmic, pyridoxal-5′-phosphate-dependent amino acid racemase in Pseudomonas taetrolens by Daisuke Matsui; Tadao Oikawa; Noriaki Arakawa; Shintaro Osumi; Frank Lausberg; Norma Stäbler; Roland Freudl; Lothar Eggeling (pp. 1045-1054).

The pyridoxal-5′-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.

Keywords: Racemase specificity; Sec pathway; Alanine racemase; Lysine racemase; Tat-translocation; Periplasmic localization

A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli by Paulina Ducka; Ulrich Eckhard; Esther Schönauer; Stefan Kofler; Gerhard Gottschalk; Hans Brandstetter; Dorota Nüss (pp. 1055-1065).

Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.

Keywords: Clostridial collagenases; Expression; Purification; Platform

Differences in the substrate specificity of glycosyltransferases involved in landomycins A and E biosynthesis by Annette Erb; Christine Krauth; Andriy Luzhetskyy; Andreas Bechthold (pp. 1067-1076).

A lanGT4 mutant of the landomycin A producer Streptomyces cyanogenus S136 was constructed, leading to the production of landomycin D with two deoxy sugars in the side chain and proving that LanGT4 is responsible for attaching the third deoxy sugar of the hexasaccharide side chain. Heterologous expression of lndGT4 of the landomycin E producer Streptomyces globisporus 1912 in the lanGT4 mutant restored landomycin A production, indicating that LndGT4, like LanGT4, also has the ability to work iteratively. A S. cyanogenus S136 mutant with a mutation in lanGT1, encoding a d-olivosyltransferase, was shown to produce landomycin I with one deoxy sugar and, surprisingly, a new landomycin derivative (landomycin L) containing a d-olivose followed by an l-rhodinose. Heterologous expression of lndGT1 of S. globisporus 1912 in the lanGT1 mutant did not restore landomycin A production but led to the formation of a second new landomycin derivative (landomycin K) containing an unusual pentasaccharide chain (d-olivose–d-olivose–l-rhodinose–d-olivose–l-rhodinose). The formation of landomycin L and landomycin K is most probably attributed to the high substrate flexibility of the rhodinosyltransferase LanGT4.

Keywords: Streptomyces ; Combinatorial biosynthesis; Glycosyltransferase; Secondary metabolite

Biological analysis of the deletion mutants of Staphylococcal enterotoxin C2 by Xiaogang Wang; Huiwen Zhang; Mingkai Xu; Changxiao Liu; Chenggang Zhang (pp. 1077-1084).

To investigate the functional domains involved in the biological activity of staphylococcal enterotoxin (SEC2), a series of SEC2 mutants were constructed. Deletion of the last 77 amino acids at the C-terminus of SEC2 did not affect its native superantigen and fever activities, and further removal of the C-terminal residues reduced SEC2 activities significantly. On the other hand, the mutants lacking 18 or more N-terminal residues severely impaired superantigen activity. These data indicated that the functional regions for the biological activities of SEC2 were confined to N-terminal domain, further implied that the proper three-dimensional structure of SEC2 is not needed for its biological activities. Our results deliver valuable information that it is possible to design new SEC2 immunotherapeutic agents which have the superantigen activity and low molecular weight for permeability.

Keywords: Staphylococcal enterotoxin C2; Superantigen activity; Fever activity; Immunotherapeutic agent

Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8 by Gang-hua Lang; Naoto Ogawa (pp. 1085-1094).

TfdT is a LysR-type transcriptional regulator that activates the transcription of the chlorocatechol degradative gene operon tfdCDEF of the chlorobenzoate-degrading bacterium Burkholderia sp. NK8. To identify the amino acids involved in the effector recognition by TfdT, a polymerase-chain-reaction-based random mutagenesis protocol was applied to introduce mutations into the tfdT gene. Nine types of TfdT mutant bearing a single-amino-acid substitution at positions, Lys-129, Arg-199, Val-226, Val-246, and Pro-267 were obtained on the basis of their altered effector profiles and enhanced responses particularly to 2-chlorobenzoate, 2-aminobenzoate, and 2,6-dichlorobenzoate. All the TfdT mutants showed enhanced response to the effectors with a chloro-group in C-2 of benzoic acid. A homology model of wild-type TfdT was built on the basis of the crystal structure of CbnR with SwissModel. In this model, residues corresponding to the mutation sites of isolated TfdT mutants were located at the interface between the domains RD-I and RD-II. The findings that these TfdT mutants expressed altered effector specificities and enhanced responses to specific effectors suggest that these five residues are involved in effector binding by TfdT.

Keywords: LysR-type transcriptional regulator; Chlorocatechol ortho-cleavage pathway; TfdT; Random mutagenesis; Burkholderia sp. NK8; Chlorobenzoate

Structural understanding of quorum-sensing inhibitors by molecular modeling study in Pseudomonas aeruginosa by Cheoljin Kim; Jaeeun Kim; Hyung-Yeon Park; Joon-Hee Lee; Hee-Jin Park; Chan Kyung Kim; Jeyong Yoon (pp. 1095-1103).

Inhibitors of 3OC12, an initial signal molecule of the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa have been developed. Eight inhibitor candidates were synthesized by substituting the head part of 3-oxododecanoyl-homoserine lactone (3OC12) with different aromatic rings, and their docking poses and scores (binding energies) were predicted by in silico modeling study. All compounds gave better docking scores than 3OC12 and good inhibition effects on LasR activity in the in vivo bioassay. Like the modifications in the tail part of 3OC12 in our previous study Kim et al. (2008), the head-part modifications also showed inhibition activity in a fairly good proportion to the docking scores from the modeling analysis. This implies that the head part of 3OC12 also contributes significantly to forming the active conformation of the LasR-3OC12 complex, and its modification could effectively induce the inactive conformation of the complex. We suggest that the head part of 3OC12 is also a good target moiety to develop the structure-based Pseudomonas QS inhibitors.

Keywords: Quorum sensing; Quorum-sensing inhibitor; Autoinducer; In silico modeling; FlexX docking; Docking scores; Pseudomonas aeruginosa

Optimization of l-methionine feeding strategy for improving S-adenosyl-l-methionine production by methionine adenosyltransferase overexpressed Pichia pastoris by Hui Hu; Jiangchao Qian; Ju Chu; Yonghong Wang; Yingping Zhuang; Siliang Zhang (pp. 1105-1114).

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.

Keywords: S-adenosyl-l-methionine; Methionine adenosyltransferase; l-methionine; Pichia pastoris ; ATP

Molecular characterization of native isolates of lactic acid bacteria, bifidobacteria and yeasts for beneficial attributes by Arekal N. Roopashri; Mandyam C. Varadaraj (pp. 1115-1126).

In a changing scenario of food habits being associated with wellness factors through the concepts of probiotics and prebiotics, an attempt has been made to characterize on molecular basis, the desirable benefits associated with natural isolates of lactic acid bacteria, bifidobacteria, and yeasts. From a diverse range of foods and related samples, based on conventional microbiological protocols, three well-characterized natural isolates of Lactobacillus plantarum MTCC 5422, Bifidobacterium adolescentis MTCC 5423 and Saccharomyces cerevisiae MTCC 5421 were selected. The cultures of L. plantarum and B. adolescentis showed positive polymerase chain reaction (PCR) amplification with oligonucleotide primers targeting genus-specific 16 S rRNA for Lactobacillus and fructose-6-phosphate phosphoketolase for Bifidobacterium. Similarly, species-specific positive amplification in PCR was observed with primers of phytase (acid phosphatase) in S. cerevisiae and α-d-galactosidase and bile salt hydrolase in L. plantarum and B. adolescentis. The cultures of L. plantarum and B. adolescentis exhibited a broad spectrum antibacterial activity against selected foodborne pathogenic bacterial species and tolerance to acid and bile. Gene sequence of respective PCR-amplified products confirmed the genetic identity of the isolated cultures as L. plantarum and B. adolescentis showing 99% homology with the documented sequence of established gene bank.

Keywords: Lactobacillus plantarum ; Bifidobacterium adolescentis ; Saccharomyces cerevisiae ; Probiotic; Prebiotic; Molecular characterization; Phylogeny

Antifungal potential of essential oil and various organic extracts of Nandina domestica Thunb. against skin infectious fungal pathogens by Vivek K. Bajpai; Jung In Yoon; Sun Chul Kang (pp. 1127-1133).

This study was undertaken to assess the in vitro antifungal potential of the essential oil and n-hexane, chloroform, ethyl acetate, and methanol extracts of Nandina domestica Thunb. against dermatophytes, the casual agents of superficial infections in animals and human beings. The oil (1,000 μg/disc) and extracts (1,500 μg/disc) revealed 31.1–68.6% and 19.2–55.1% antidermatophytic effect against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, Trichophyton mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348, and M. canis KCTC 6349, respectively, along with their respective minimum inhibitory concentration values ranging from 62.5 to 500 and 125 to 2,000 μg/ml. Also, the oil had strong detrimental effect on spore germination of all the tested dermatophytic fungi as well as concentration and time-dependent kinetic inhibition of T. rubrum KCTC 6375. The present results demonstrated that N. domestica mediated oil and extracts could be potential sources of natural fungicides to control certain important dermatophytic fungi.

Keywords: Nandina domestica Thunb.; Antifungal potential; Essential oil; Organic extracts; Skin infectious fungal pathogens

Application of quantitative RT-PCR to determine the distribution of Microthrix parvicella in full-scale activated sludge treatment systems by S. K. Sheena Kumari; Z. Marrengane; F. Bux (pp. 1135-1141).

Three wastewater treatment plants in South Africa were investigated to understand the phylogeny and distribution of Microthrix parvicella using real-time polymerase chain reaction (RT-PCR). The phylogenetic analysis of the 16S rRNA of M. parvicella revealed 98% to 100% homology of South African clones to M. parvicella reported in Genbank. The standard curves for RT-PCR showed R2 values greater than 0.99, accurate for quantification. The relative occurrence of M. parvicella 16S rRNA gene copies in the three wastewater treatment plants was in the range 0% to 3.97%. M. parvicella copies increased when the environmental temperature (≤20°C) and food/microorganism (F/M) ratio was low. The M. parvicella 16S rRNA copies could be positively correlated to the sludge volume index at low temperature. At higher temperature, there was a rapid reduction in M. parvicella population irrespective of other favorable factors, indicating the strong influence of temperature on filamentous proliferation. RT-PCR has potential applications in wastewater treatment plants to monitor sudden shift in the microbial population and assessing the plants efficacy.

Keywords: Microthrix parvicella ; 16S rRNA analysis; Real-time polymerase chain reaction; Food/microorganism ratio; Sludge volume index

Microbial response to reinjection of produced water in an oil reservoir by Kristine Lysnes; Gunhild Bødtker; Terje Torsvik; Eva Ø. Bjørnestad; Egil Sunde (pp. 1143-1157).

The microbial response to produced water reinjection (PWRI) in a North Sea oil field was investigated by a combination of cultivation and culture-independent molecular phylogenetic techniques. Special emphasise was put on the relationship between sulphate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB), and results were used to evaluate the possibility of nitrate treatment as a souring management tool during PWRI. Samples were collected by reversing the flow of the injection water, which provided samples from around the injection area. The backflowed samples were compared to produced water from the same platform and to backflowed samples from a biocide-treated seawater injector, which was the previous injection water treatment of the PWRI well. Results showed that reinjection of produced water promoted growth of thermophilic SRB. Thermophilic fatty acid oxidising NRB and potential nitrate-reducing sulphide-oxidising bacteria were also found. The finding of thermophilic NRB makes nitrate treatment during PWRI possible, although higher nitrate concentration will be necessary to compensate for the increased SRB activity.

Keywords: Oil reservoir; Produced water; Reinjection; SRB; NRB; NR-SOB

Kinetic analysis on the two-step processes of AOB and NOB in aerobic nitrifying granules by Fang Fang; Bing-Jie Ni; Xiao-Yan Li; Guo-Ping Sheng; Han-Qing Yu (pp. 1159-1169).

Complete granulation of nitrifying sludge was achieved in a sequencing batch reactor. For the granular sludge, batch experiments were conducted to characterize the kinetic features of ammonia oxidizers (AOB) and nitrite oxidizers (NOB) in the granules using the respirometric method. A two-step nitrification model was established to determine the kinetic parameters of both AOB and NOB. In addition to nitrification reactions, the new model also took into account biomass maintenance and mass transfer through the granules. The yield coefficient, maximum specific growth rate, and affinity constant for ammonium for AOB were 0.21 g chemical oxygen demand (COD) g⁻¹ N, 0.09 h⁻¹, and 9.1 mg N L⁻¹, respectively, whereas the corresponding values for NOB were 0.05 g COD g⁻¹ N, 0.11 h⁻¹, and 4.85 mg N L⁻¹, respectively. The model developed in this study performed well in simulating the oxygen uptake rate and nitrogen conversion kinetics and in predicting the oxygen consumption of the AOB and NOB in aerobic granules.

Keywords: Aerobic granule; Ammonia oxidizers (AOB); Biological wastewater treatment; Nitrification; Nitrite oxidizers (NOB); Kinetics

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy by Christophe Sandt; Truis Smith-Palmer; Jonathan Comeau; David Pink (pp. 1171-1182).

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.

Keywords: Confocal Raman microscopy; Biomass; Hydration; Bacteria; Map

Effect of emodin on Candida albicans growth investigated by microcalorimetry combined with chemometric analysis by W. J. Kong; J. B. Wang; C. Jin; Y. L. Zhao; C. M. Dai; X. H. Xiao; Z. L. Li (pp. 1183-1190).

Using the 3114/3115 thermal activity monitor (TAM) air isothermal microcalorimeter, ampoule mode, the heat output of Candida albicans growth at 37°C was measured, and the effect of emodin on C. albicans growth was evaluated by microcalorimetry coupled with chemometric methods. The similarities between the heat flow power (HFP)–time curves of C. albicans growth affected by different concentrations of emodin were calculated by similarity analysis (SA). In the correspondence analysis (CA) diagram of eight quantitative parameters taken from the HFP–time curves, it could be deduced that emodin had definite dose-effect relationship as the distance between different concentrations of it increased along with the dosage and the effect. From the principal component analysis (PCA) on eight quantitative parameters, the action of emodin on C. albicans growth could be easily evaluated by analyzing the change of values of the main two parameters, growth rate constant k ₂ and maximum power output $$ P_{ ext{m}}^2 $$ . The coherent results of SA, CA, and PCA showed that emodin at different concentrations had different effects on C. albicans growth metabolism: A low concentration (0–10 μg ml⁻¹) poorly inhibited the growth of C. albicans, and a high concentration (15–35 μg ml⁻¹) could notably inhibit growth of this fungus. This work provided a useful idea of the combination of microcalorimetry and chemometric analysis for investigating the effect of drug and other compounds on microbes.

Keywords: Emodin; Candida albicans ; Microcalorimetry; Chemometric analysis

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Applied Microbiology and Biotechnology (v.83, #6)