Applied Biochemistry and Biotechnology (v.181, #1)

Microbial Biofertilizer Decreases Nicotine Content by Improving Soil Nitrogen Supply by Cui Shang; Anwei Chen; Guiqiu Chen; Huanke Li; Song Guan; Jianmin He (1-14).
Biofertilizers have been widely used in many countries for their benefit to soil biological and physicochemical properties. A new microbial biofertilizer containing Phanerochaete chrysosporium and Bacillus thuringiensis was prepared to decrease nicotine content in tobacco leaves by regulating soil nitrogen supply. Soil NO3 -N, NH4 +-N, nitrogen supply-related enzyme activities, and nitrogen accumulation in plant leaves throughout the growing period were investigated to explore the mechanism of nicotine reduction. The experimental results indicated that biofertilizer can reduce the nicotine content in tobacco leaves, with a maximum decrement of 16–18 % in mature upper leaves. In the meantime, the total nitrogen in mature lower and middle leaves increased with the application of biofertilizer, while an opposite result was observed in upper leaves. Protein concentration in leaves had similar fluctuation to that of total nitrogen in response to biofertilizer. NO3 -N content and nitrate reductase activity in biofertilizer-amended soil increased by 92.3 and 42.2 %, respectively, compared to those in the control, whereas the NH4 +-N and urease activity decreased by 37.8 and 29.3 %, respectively. Nitrogen uptake was improved in the early growing stage, but this phenomenon was not observed during the late growth period. Nicotine decrease is attributing to the adjustment of biofertilizer in soil nitrogen supply and its uptake in tobacco, which result in changes of nitrogen content as well as its distribution in tobacco leaves. The application of biofertilizer containing P. chrysosporium and B. thuringiensis can reduce the nicotine content and improve tobacco quality, which may provide some useful information for tobacco cultivation.
Keywords: Biofertilizer; Nicotine; Phanerochaete chrysosporium ; Bacillus thuringiensis ; Tobacco; Nitrogen

Effects of Melatonin on Colchicine-Treated PLBs of Dendrobium sonia-28 Orchid by M . S. Lim; J. J. J. Antony; S. M. Shahinul Islam; Z . Suhana; S. Sreeramanan (15-31).
Dendrobium hybrid orchid is popular in orchid commercial industry due to its short life cycle and ability to produce various types of flower colours. This study was conducted to identify the morphological, biochemical and scanning electron microscopy (SEM) analysis in the Dendrobium sonia-28 orchid plants. In this study, 0.05 and 0.075 % of colchicine-treated Dendrobium sonia-28 (4-week-old culture) protocorm-like bodies (PLBs) were treated in different concentrations of melatonin (MEL) posttreatments (0, 0.05, 0.1, 0.5, 1, 5 and 10 μM). Morphological parameters such as number of shoots, growth index and number of PLBs were determined. In the 0.05 and 0.075 % of colchicine-treated PLBs which were posttreated with 0.05 μM MEL resulted in the highest value of the morphological parameters tested based on the number of shoots (84.5 and 96.67), growth index (16.94 and 12.15) and number of PLBs (126.5 and 162.33), respectively. SEM analysis of the 0.05 μM MEL posttreatment on both the colchicine-treated regenerated PLBs showed irregular cell lineages, and some damages occurred on the stomata. This condition might be due to the effect of plasmolyzing occurred in the cell causing irregular cell lineages.
Keywords: Dendrobium sonia-28; Protocorm-like bodies; Melatonin; Scanning electron microscopy

Variation in the Gut Microbiota of Termites (Tsaitermes ampliceps) Against Different Diets by Lijuan Su; Lele Yang; Shi Huang; Yan Li; Xiaoquan Su; Fengqin Wang; Cunpei Bo; En Tao Wang; Andong Song (32-47).
Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1–V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota.
Keywords: Tsaitermes ampliceps ; Gut symbionts; Lignocellulose degradation; Corn stover; Filter paper; 454 Pyrosequencing

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC50 values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da.
Keywords: Fermentation; Bioactive peptides; Size exclusion chromatography; MALDI-TOF

Two novel sugar acid-binding lectins were purified from Haplomitrium mnioides (Lindb.) Schust. using a procedure consisting of ammonium sulfate precipitation, G-50 gel filtration, hydroxyapatite chromatography, and HW-50 gel filtration. We reported their partial physicochemical properties: molecular weight, affinity for carbohydrates and organic acids, pH stability, and dependence of their hemagglutination activity on metal ions. We also determined their N-terminal amino acid sequences. H. mnioides lectins (HMLs) were monomers (one with a molecular weight of approximately 27 kDa, and the other with a molecular weight of approximately 105 kDa) under both nonreducing and reducing conditions. They were named HML27 and HML105, respectively. Both HMLs had an affinity for N-acetylneuraminic acid, d-glucuronic acid, d-glucaric acid, bovine submaxillary mucin, heparin, and organic acids, such as citrate, 2-oxoglutaric acid, and d-2-hydroxyglutarate. Furthermore, HML27 had an affinity for α-d-galacturonic acid, d-malate, l-malate, and pyruvate, while HML105 had an affinity for d-gluconic acid. HML27 and HML105 are novel plant lectins: they have an affinity for sugar acids and organic acids and specifically recognize the carboxyl group, and there is no homology between their N-terminal amino acid sequences and those of the previously described lectins and agglutinins.
Keywords: Bryophyte; Haplomitrium mnioides ; Organic acids; Plant lectin; Purification; Sugar acids

Modulating Mobility: a Paradigm for Protein Engineering? by Margaret McAuley; David J. Timson (83-90).
Proteins are highly mobile structures. In addition to gross conformational changes occurring on, for example, ligand binding, they are also subject to constant thermal motion. The mobility of a protein varies through its structure and can be modulated by ligand binding and other events. It is becoming increasingly clear that this mobility plays an important role in key functions of proteins including catalysis, allostery, cooperativity, and regulation. Thus, in addition to an optimum structure, proteins most likely also require an optimal dynamic state. Alteration of this dynamic state through protein engineering will affect protein function. A dramatic example of this is seen in some inherited metabolic diseases where alternation of residues distant from the active site affects the mobility of the protein and impairs function. We postulate that using molecular dynamics simulations, experimental data or a combination of the two, it should be possible to engineer the mobility of active sites. This may be useful in, for example, increasing the promiscuity of enzymes. Thus, a paradigm for protein engineering is suggested in which the mobility of the active site is rationally modified. This might be combined with more “traditional” approaches such as altering functional groups in the active site.
Keywords: Enzyme engineering; Molecular dynamics; Protein flexibility; Active site; Biocatalysis; Conformational change

Methane Potential and Microbial Community Dynamics in Anaerobic Digestion of Silage and Dry Cornstalks: a Substrate Exchange Study by Ye Zhao; Xufeng Yuan; Boting Wen; Xiaofen Wang; Wanbin Zhu; Zongjun Cui (91-111).
Silage and dry are the two typical cornstalk forms. Either form could be used as substrate in biogas plants and might be replaced by another when shortage occurred. This study focused on the feeding sequence of these two kinds of feedstocks, aiming to discuss their specific methane potential (SMP). A 15-day hydraulic retention time was chosen for semi-continuous experiments based on the batch test results. In semi-continuous experiments, before and after feedstocks were exchanged, the significantly decreased and comparable SMPs of silage and dry cornstalks indicated that a basis of unstable digestion would result in incomplete methane release from the subsequent digestion. A higher similarity of bacterial community structure and greater quantity of bacteria were shown in acidified silage cornstalk digestion through band similarity analysis. Methanosaetaceae and methanomicrobiales were the predominant methanogens, and aceticlastic methanogenesis was the main route for methane production. The different feeding sequences affected the hydrolysis course and further influenced the methanogenic proliferation. Our work suggests that silage cornstalk digestion should be conducted before dry cornstalk digestion.
Keywords: Silage cornstalk; Specific methane potential; Anaerobic digestion; Feeding sequence; Methanogens

Harvesting Microalgae with Different Sources of Starch-Based Cationic Flocculants by Chengrong Peng; Shuangshuang Li; Jiaoli Zheng; Shun Huang; Dunhai Li (112-124).
In this study, starches obtained from wheat, potato, and corn were used to synthesize cationic starches (CS), and the flocculation efficiency of these materials was tested with Chlorella pyrenoidosa and Botryococcus braunii cultures under different conditions. Our results indicated that these three CS had differing degrees of substitution following identical synthesis conditions. The various CS functioned similarly in this study, and the desired harmless flocculation efficiency was obtained at low dosages, with CS to microalgal biomass ratios of approximately 89 and 119 mg g−1 for C. pyrenoidosa and B. braunii, respectively. Impressive harmless harvesting efficiencies were obtained at lower dosages with respect to appropriate stirring time before the settling, with ratios ranging from 58 to 78 mg g−1 for C. pyrenoidosa cultures. The cost of microalgae harvesting can be cut dramatically by choosing cheaper starches prior to the synthetic CS and by applying suitable flocculation procedures.
Keywords: Cationic starch; Flocculation procedures; Oleaginous microalgae; Biomass harvest; Harmless

Thermophilic dry methane fermentation is advantageous for feedstock with high solid content. Distillation residue with 65.1 % moisture content was eluted from ethanol fermentation of kitchen waste and subjected to thermophilic dry methane fermentation, after adjusting the moisture content to 75 %. The effect of carbon to nitrogen (C/N) ratio on thermophilic dry methane fermentation was investigated. Results showed that thermophilic dry methane fermentation could not be stably performed for >10 weeks at a C/N ratio of 12.6 and a volatile total solid (VTS) loading rate of 1 g/kg sludge/d; however, it was stably performed at a C/N ratio of 19.8 and a VTS loading rate of 3 g/kg sludge/d with 83.4 % energy recovery efficiency. Quantitative PCR analysis revealed that the number of bacteria and archaea decreased by two orders of magnitude at a C/N ratio of 12.6, whereas they were not influenced at a C/N ratio of 19.8. Microbial community analysis revealed that the relative abundance of protein-degrading bacteria increased and that of organic acid-oxidizing bacteria and acetic acid-oxidizing bacteria decreased at a C/N ratio of 12.6. Therefore, there was accumulation of NH4 + and acetic acid, which inhibited thermophilic dry methane fermentation.
Keywords: Anaerobic digestion; Ethanol fermentation; Dry methane fermentation; Kitchen waste; Stillage

Multi-phase anaerobic reactor for H2 and CH4 production from paperboard mill wastewater was studied. The reactor was operated at hydraulic retention times (HRTs) of 12, 18, 24, and 36 h, and organic loading rates (OLRs) of 2.2, 1.5, 1.1, and 0.75 kg chemical oxygen demand (COD)/m3 day, respectively. HRT of 12 h and OLR of 2.2 kg COD/m3 day provided maximum hydrogen yield of 42.76 ± 14.5 ml/g CODremoved and volumetric substrate uptake rate (−rS) of 16.51 ± 4.43 mg COD/L h. This corresponded to the highest soluble COD/total COD (SCOD/TCOD) ratio of 56.25 ± 3.3 % and the maximum volatile fatty acid (VFA) yield (YVFA) of 0.21 ± 0.03 g VFA/g COD, confirming that H2 was mainly produced through SCOD conversion. The highest methane yield (18.78 ± 3.8 ml/g CODremoved) and −rS of 21.74 ± 1.34 mgCOD/L h were achieved at an HRT of 36 h and OLR of 0.75 kg COD/m3 day. The maximum hydrogen production rate (HPR) and methane production rate (MPR) were achieved at carbon to nitrogen (C/N) ratio of 47.9 and 14.3, respectively. This implies the important effect of C/N ratio on the distinction between the dominant microorganism bioactivities responsible for H2 and CH4 production.
Keywords: Paperboard mill wastewater; Anaerobic baffled reactor; Operating conditions; Hydrogen; Methane; Heating energy consumption

In this study, single-stage and two-phase semi-continuous thermophilic anaerobic reactors fed with diluted (3 % total solids (TS) and 1.8 % volatile solids (VS)) chicken manure at three different hydraulic retention times (HRTs) were compared interms of biogas production rate, methane content of the produced biogas, and VS and TS removal. Along the study, HRTs of 16, 12, and 8 days were implemented to the single-stage and the two-phase systems. It was observed that the single-stage anaerobic system was superior to the two-phase anaerobic system according to their biogas production rates (517 vs. 356, 551 vs. 359, 459 vs. 386 (mL/g VSfeed)) at all HRTs. On the other hand, methane content of the biogas produced was higher in the two-phase system compared to the single-stage system.
Keywords: Anaerobic treatment; Chicken manure; Single stage; Two phase; Biogas production

Improvement and Characterization in Enzymatic Hydrolysis of Regenerated Wheat Straw Dissolved by LiCl/DMAc Solvent System by Gaoxiang Qi; Lian Xiong; Bo Wang; Xiaoqing Lin; Hairong Zhang; Hailong Li; Chao Huang; Xuefang Chen; Can Wang; Xinde Chen (177-191).
Lithium chloride (LiCl)/N,N-dimethylacetamide (DMAc) solvent system was used to dissolve native and pretreated wheat straw materials in order to promote the enzymatic hydrolysis process. The dissolution ratio of wheat straw in LiCl/DMAc solvent system increased when dilute sulfuric acid or ethanol-sulfuric acid mixture pretreatment was conducted before dissolution. The materials regenerated from LiCl/DMAc solution exhibited obvious changes in structure and morphology, as revealed by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. The cellulose and xylan digestibilities of regenerated materials were improved obviously. The cellulose digestibilities of materials regenerated from native and pretreated wheat straw by dilute sulfuric acid and ethanol-sulfuric acid mixture were 61.8, 81.2, and 84.4 %, respectively, with 25 FPU of cellulase and 187 IU of xylanase after 96 h. These values were significantly higher than the digestibilities of 0, 33.2, and 57.5 % obtained from corresponding materials before dissolution. Dissolution treatment of wheat straw by LiCl/DMAc solvent system provides an alternative method for efficient enzymatic hydrolysis.
Keywords: Wheat straw; Pretreatment; LiCl/DMAc solvent system; Dissolution and regeneration; Enzymatic hydrolysis

Quorum sensing (QS) plays an important role in the biofilm formation, production of virulence factors and stress responses in Vibrio harveyi. Therefore, interrupting QS is a possible approach to modulate bacterial behavior. In the present study, three docking protocols, such as Rigid Receptor Docking (RRD), Induced Fit Docking (IFD), and Quantum Polarized Ligand Docking (QPLD) were used to elucidate the binding mode of boronic acid derivatives into the binding pocket of LuxP protein in V. harveyi. Among the three docking protocols, IFD accurately predicted the correct binding mode of the studied inhibitors. Molecular dynamics (MD) simulations of the protein-ligand complexes indicates that the inter-molecular hydrogen bonds formed between the protein and ligand complex remains stable during the simulation time. Pharmacophore and shape-based virtual screening were performed to find selective and potent compounds from ChemBridge database. Five hit compounds were selected and subjected to IFD and MD simulations to validate the binding mode. In addition, enrichment calculation was performed to discriminate and separate active compounds from the inactive compounds. Based on the computational studies, the potent Bicyclo [2.2.1] hept-5-ene-2,3-dicarboxylic acid-2,6-dimethylpyridine 1-oxide (ChemBridge_5144368) was selected for in vitro assays. The compound exhibited dose dependent inhibition in bioluminescence and also inhibits biofilm formation in V. harveyi to the level of 64.25 %. The result from the study suggests that ChemBridge_5144368 could serve as an anti-quorum sensing molecule for V. harveyi.
Keywords: Quorum sensing; Boronic acid derivatives; Molecular docking; Molecular dynamics simulations; In vitro assays

A Novel Heptapeptide with Tyrosinase Inhibitory Activity Identified from a Phage Display Library by Huali Nie; Lin Liu; Huiqin Yang; Hongzhen Guo; Xiang Liu; Yuanhao Tan; Wen Wang; Jing Quan; Limin Zhu (219-232).
Peptidic inhibition of the enzyme tyrosinase, responsible for skin pigmentation and food browning, would be extremely useful for the food, cosmetics, and pharmaceutical industries. In order to identify novel inhibitory peptides, a library of short sequence oligopeptides was screened to reveal direct interaction with the tyrosinase. A phage displaying heptapeptide (IQSPHFF) was found to bind most strongly to tyrosinase. The inhibitory activity of the heptapeptide was evaluated using mushroom tyrosinase. The results showed that the peptide inhibited both the monophenolase and diphenolase activities of mushroom tyrosinase with IC50 values of 1.7 and 4.0 mM, respectively. The heptapeptide is thought to be a reversible competitive inhibitor of diphenolase with the inhibition constants (Ki) of 0.765 mM. To further investigate how the heptapeptide exerts its inhibitory effect, a docking study between tyrosinase and heptapeptide was performed. The simulation showed that the heptapeptide binds in the active site of the enzyme near the catalytically active Cu ions and forms hydrogen bonds with five histidine residues on the active site. Phage display technology is thus a useful approach for the screening of potential tyrosinase inhibitors and could be widely applicable to a much wider range of enzymes.
Keywords: Mushroom tyrosinase; Inhibition; Heptapeptide; Phage display; Molecular docking

The Protein/Peptide Direct Virus Inactivation During Chromatographic Process: Developing Approaches by Georgii L. Volkov; Sergiy P. Havryliuk; Ievgenia M. Krasnobryzha; Olena S. Havryliuk (233-249).
Virus clearance is required for pharmaceutical preparations derived from animal or human sources such as blood products, vaccines, recombinant proteins produced in mammalian cell lines, etc. High cost and substantial protein losses during virus inactivation are significant problems for protein/peptide manufacturing. The goal of this project was to develop a method to perform virus inactivation in a course of protein chromatographic purification. Another goal was to show that the chromatographic adsorbent can serve as reliable “sieva” for mechanical washing away of infecting viruses. Using chromatographic, photometric, IFA, and RT-PCR approaches, it was discovered that high temperature-depending dynamic capacity of adsorbent allowed to perform a virus inactivation directly in a chromatographic column by solvent/detergent treatment. The peptide/protein biological activity was completely preserved. Using this new approach enveloped and nonenveloped viruses were effectively removed protein preparation. In addition, it was shown that RT-PCR method demonstrates more precise and reproducible results and robust properties for assessment of virus reduction than virus titer followed by infectivity studies. Presented method allowed to obtain the factor of virus concentration decrease (FVD) values that were higher than those provided by known technologies and was sufficient for a full inactivation of viruses. The method is recommended to use in pharmaceutical industry.
Keywords: Protien; Peptide; Virus inactivation; Chromatography; Dynamic capacity; Virus titer; Infectivity; RT-PCR

Numerical Simulation of Mass Transfer and Three-Dimensional Fabrication of Tissue-Engineered Cartilages Based on Chitosan/Gelatin Hybrid Hydrogel Scaffold in a Rotating Bioreactor by Yanxia Zhu; Kedong Song; Siyu Jiang; Jinglian Chen; Lingzhi Tang; Siyuan Li; Jiangli Fan; Yiwei Wang; Jiaquan Zhao; Tianqing Liu (250-266).
Cartilage tissue engineering is believed to provide effective cartilage repair post-injuries or diseases. Biomedical materials play a key role in achieving successful culture and fabrication of cartilage. The physical properties of a chitosan/gelatin hybrid hydrogel scaffold make it an ideal cartilage biomimetic material. In this study, a chitosan/gelatin hybrid hydrogel was chosen to fabricate a tissue-engineered cartilage in vitro by inoculating human adipose-derived stem cells (ADSCs) at both dynamic and traditional static culture conditions. A bioreactor that provides a dynamic culture condition has received greater applications in tissue engineering due to its optimal mass transfer efficiency and its ability to simulate an equivalent physical environment compared to human body. In this study, prior to cell-scaffold fabrication experiment, mathematical simulations were confirmed with a mass transfer of glucose and TGF-β2 both in rotating wall vessel bioreactor (RWVB) and static culture conditions in early stage of culture via computational fluid dynamic (CFD) method. To further investigate the feasibility of the mass transfer efficiency of the bioreactor, this RWVB was adopted to fabricate three-dimensional cell-hydrogel cartilage constructs in a dynamic environment. The results showed that the mass transfer efficiency of RWVB was faster in achieving a final equilibrium compared to culture in static culture conditions. ADSCs culturing in RWVB expanded three times more compared to that in static condition over 10 days. Induced cell cultivation in a dynamic RWVB showed extensive expression of extracellular matrix, while the cell distribution was found much more uniformly distributing with full infiltration of extracellular matrix inside the porous scaffold. The increased mass transfer efficiency of glucose and TGF-β2 from RWVB promoted cellular proliferation and chondrogenic differentiation of ADSCs inside chitosan/gelatin hybrid hydrogel scaffolds. The improved mass transfer also accelerated a dynamic fabrication of cell-hydrogel constructs, providing an alternative method in tissue engineering cartilage.
Keywords: Chitosan/gelatin hybrid hydrogel; Rotating wall vessel bioreactor; Static culture; Mass transfer efficiency; Tissue-engineered cartilage

Population Genetic Structure and Marker Trait Associations Using Morphological, Phytochemical and Molecular Parameters in Habenaria edgeworthii—a Threatened Medicinal Orchid of West Himalaya, India by Lalit Giri; Arun Kumar Jugran; Amit Bahukhandi; Praveen Dhyani; Indra D. Bhatt; Ranbeer Singh Rawal; Shyamal Kumar Nandi; Uppeandra Dhar (267-282).
Habenaria edgeworthii Hook. f. ex Collett is an important terrestrial orchid used in different Ayurvedic formulations. In the present study, variations among morphological, phytochemical and molecular markers were assessed. A significant difference was observed among populations using morphological traits. Inter-simple sequence repeat (ISSR) data revealed lower genetic diversity at population level (He = 0.207) as compared to species level (He = 0.334). Analysis of molecular variance (AMOVA) indicates 74 % variation among populations and 26 % within population. Tuber extracts showed significantly (p < 0.05) higher total phenolics and flavonoids among the populations. Antioxidant activity determined by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays exhibited considerable antioxidant potential. Furthermore, the associations between molecular and morphological and phytochemical attributes were studied using multiple regression analysis (MRA). Several ISSR fragments were associated with some morphological and phytochemical traits. These ISSR fragments can be useful for breeding programme of the species when no other genetic information, such as linkage maps and quantitative trait loci, is available.
Keywords: Phytochemical; ISSR analysis; Medicinal plant; Trait association

Antioxidant Activities of Peptoid-Grafted Chitosan Films by P-H. Elchinger; C. Delattre; S. Faure; O. Roy; S. Badel; T. Bernardi; P. Michaud; C. Taillefumier (283-293).
The aim of this study was to investigate the possibility of immobilizing peptoid on chitosan film in order to generate new active material. Chitosan films have been grafted for the first time with short-length peptoid oligomers displaying antioxidant activities. The antioxidant activity of the selected peptoids was initially investigated with the DPPH assay and hydroxyl radical procedure. The metal chelating capacity of peptoids was also evaluated prior to their covalent attachment to chitosan. The benefit of chitosan functionalization with respect to its intrinsic antioxidant properties was finally evaluated in the present study. Interestingly, an increase of up to 90 % of the antioxidant activity of chitosan was observed.
Keywords: Antioxidant; Chitosan film; Grafting; Biosourced material; Peptoid; Polysaccharide

Creating Economic Incentives for Waste Disposal in Developing Countries Using the MixAlco Process by Sagar Lonkar; Zhihong Fu; Melinda Wales; Mark Holtzapple (294-308).
In rapidly growing developing countries, waste disposal is a major challenge. Current waste disposal methods (e.g., landfills and sewage treatment) incur costs and often are not employed; thus, wastes accumulate in the environment. To address this challenge, it is advantageous to create economic incentives to collect and process wastes. One approach is the MixAlco process, which uses methane-inhibited anaerobic fermentation to convert waste biomass into carboxylate salts, which are chemically converted to industrial chemicals and fuels. In this paper, humanure (raw human feces and urine) is explored as a possible nutrient source for fermentation. This work focuses on fermenting municipal solid waste (energy source) and humanure (nutrient source) in batch fermentations. Using the Continuum Particle Distribution Model (CPDM), the performance of continuous countercurrent fermentation was predicted at different volatile solid loading rates (VSLR) and liquid residence times (LRT). For a four-stage countercurrent fermentation system at VSLR = 4 g/(L∙day), LRT = 30 days, and solids concentration = 100 g/L liquid, the model predicts carboxylic acid concentration of 68 g/L and conversion of 78.5 %.
Keywords: The MixAlco process; Municipal solid waste; Humanure; Continuum particle distribution model

The Environmental Issues of DDT Pollution and Bioremediation: a Multidisciplinary Review by Ahlem Mansouri; Mickael Cregut; Chiraz Abbes; Marie-Jose Durand; Ahmed Landoulsi; Gerald Thouand (309-339).
DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane) is probably the best known and most useful organochlorine insecticide in the world which was used since 1945 for agricultural purposes and also for vector-borne disease control such as malaria since 1955, until its banishment in most countries by the Stockholm convention for ecologic considerations. However, the World Health Organization allowed its reintroduction only for control of vector-borne diseases in some tropical countries in 2006. Due to its physicochemical properties and specially its persistence related with a half-life up to 30 years, DDT linked to several health and social problems which are due to its accumulation in the environment and its biomagnification properties in living organisms. This manuscript compiles a multidisciplinary review to evaluate primarily (i) the worldwide contamination of DDT and (ii) its (eco) toxicological impact onto living organisms. Secondly, several ways for DDT bioremediation from contaminated environment are discussed. For this, reports on DDT biodegradation capabilities by microorganisms and ways to enhance bioremediation strategies to remove DDT are presented. The different existing strategies for DDT bioremediation are evaluated with their efficiencies and limitations to struggle efficiently this contaminant. Finally, rising new approaches and technological bottlenecks to promote DDT bioremediation are discussed.
Keywords: DDT; Pollution; Toxicity; Ecotoxicity; Biodegradation; Bioremediation

Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N2-fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.
Keywords: Co-cultivation; Cyanobacteria; Polysaccharides; Extracellular proteins; Nitrogenase activities; Rapid light curve

A stepwise pretreatment method that combines sodium hydroxide and organic acid pretreatments was proposed and investigated to maximize the recovery of main constituents of lignocellulose. The sodium hydroxide pretreatment was firstly optimized by a designed orthogonal experiment with the optimum pretreatment conditions determined as 1 wt% NaOH at 70 °C for 1 h, and 60.42 % of lignin was successfully removed during this stage. In the second stage, 0.5 % acetic acid was selected to pretreat the first-stage solid residue at 80 °C for 40 min in order to decompose hemicelluloses to soluble oligomers or monomers. Then, the whole slurry was subjected to in situ enzymatic saccharification by cellullase with a supplementation of xylanase to further degrade the xylooligosaccharides generated during the acetic acid pretreatment. The maximum reducing sugar and glucose yields achieved were 20.74 and 12.03 g/L, respectively. Furthermore, rapid ethanol fermentation and a yield of 80.3 % also testified this pretreatment method, and the in situ saccharification did not bring any negative impact on ethanol fermentation and has a broad application prospect.
Keywords: Corn stover; Sodium hydroxide; Organic acid; Pretreatment; In situ enzymatic saccharification

Interests associated with nanoparticles (NPs) are budding due to their toxicity to living species. The lethal effect of NPs depends on their nature, size, shape, and concentration. Present investigation reports that CuO NPs badly affected Brassica nigra seed germination and seedling growth parameters. However, variation in antioxidative activities and nonenzymatic oxidants is observed in plantlets. Culturing the leaf and stem explants on MS medium in presence of low concentration of CuO NPs (1–20 mg l−1) produces white thin roots with thick root hairs. These roots also show an increase in DPPH radical scavenging activity (up to 80 % at 10 mg l−1), total antioxidant, and reducing power potential (maximum in presence of 10 mg l−1 CuO NPs in the media). Nonenzymatic antioxidative molecules, phenolics and flavonoids, are observed elevated but NPs concentration dependent. We can conclude that CuO NPs can induce rooting from plant explants cultured on appropriate medium. These roots can be explored for the production of active chemical constituents.
Keywords: Antioxidative activities; Brassica nigra ; Nanoparticles; Rooting; CuO; Toxicity

Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells by Mozafar Mohammadi; Foroogh Nejatollahi; Younes Ghasemi; Sayyid Nooreddin Faraji (379-390).
Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.
Keywords: MUC18; Wnt-5a; scFv; Metastases; Molecular modeling; Protein docking

Production of Mannitol from a High Concentration of Glucose by Candida parapsilosis SK26.001 by Qing Meng; Tao Zhang; Wenting Wei; Wanmeng Mu; Ming Miao (391-406).
A novel strain, SK26.001, which can produce mannitol from a high concentration of glucose without the addition of fructose, was isolated from sugarcane juice. This strain was identified as Candida parapsilosis based on 18S ribosomal RNA (rRNA) sequence analysis and the morphological and physiological-biochemical characteristics of the strain. Under optimized fermentation conditions, the mannitol concentration in shake flasks reached 68.5 g/L. When batch fermentation was performed, the fed glucose was completely consumed after 72 h, resulting in a final mannitol concentration of 80.3 g/L. Fed-batch fermentation was then performed with glucose feed. During the fed-batch process, ammonia water was added to maintain the pH at 4.0. The mannitol concentration in the fermenter reached 97.1 g/L after 120 h, with a total glucose consumption of 284 g/L.
Keywords: Mannitol; Glucose; Candida parapsilosis ; Optimized fermentation conditions; Fed-batch fermentation

Efficiency Analysis and Mechanism Insight of that Whole-Cell Biocatalytic Production of Melibiose from Raffinose with Saccharomyces cerevisiae by Yingbiao Zhou; Yueming Zhu; Longhai Dai; Yan Men; Jinhai Wu; Juankun Zhang; Yuanxia Sun (407-423).
Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker’s yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes (suc2, mel1, and fsy1) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.
Keywords: Whole-cell biocatalysis; Saccharomyces cerevisiae ; Characteristics analysis; Melibiose; Raffinose

Based on the principle of self-coagulation of microorganisms, the flocculant-producing denitrifying bacterial TN-14 sludge was added to the continuous-flow reactor for treating domestic sewage. The bacterial TN-14 sludge acted as the main seed sludge to promote the rapid formation of aerobic granular sludge. The sludge morphology, sludge volume index (SVI) values, amounts of extracellular polymeric substances (EPS), and the role of calcium in the granulation process of the sludge were investigated. Results showed that brown aerobic granules with the particle size of 0.5 ~ 2.0 mm was successfully cultivated at 40 days, and its SVI30 decreased from 122.62 mL g−1 initially to 46.61 mL g−1 and remained at 44.28 ~ 60.51 mL g−1 afterwards. The protein (PN) content in sludge EPS increased from 76.4 mg g−1 initially to 512.3 mg g−1. Compared with PN, the polysaccharide (PS) content did not change much throughout the operation process of the bioreactor. Energy-dispersive spectrum (EDS) showed that Ca elements were deposited inside the granular sludge, and X-ray diffraction (XRD) showed that Ca elements existed in the granular sludge in the forms of CaCO3, K2CaP2O7, Ca2P2O7, and Ca4O(PO4)2. The formation mechanism of continuous-flow aerobic granular sludge was that bacterial TN-14 sludge could promote the EPS content of sludge, and PN content of EPS increases the hydrophobicity and settling performance of the sludge. Calcium mainly exists in the granular sludge in the form of inorganic calcium phosphate, and therefore plays the role of nucleation in sludge granulation.
Keywords: Aerobic granular sludge; Continuous-flow; Flocculant-producing denitrifying bacterial TN-14; Domestic sewage

Catalytic Role of Thermostable Metalloproteases from Bacillus subtilis KT004404 as Dehairing and Destaining Agent by Ramla Rehman; Maliha Ahmed; Aisha Siddique; Fariha Hasan; Abdul Hameed; Asif Jamal (434-450).
Proteases with characteristic stabilities are considered attractive candidates for industrial catalysis. In the present study, a potent bacterial strain KT004404, an inhabitant of hydrothermal vents, was isolated and characterized for protease production. Initial screening indicated that this strain produced a hydrolytic zone of 30 mm 16S rRNA-based identification revealed that our isolate was a strain of Bacillus subtilis. Optimum reaction condition for maximum protease production was determined as 55 °C, pH 6, 1 % inoculum size and malt extract as primary growth substrate supplemented with 1 % dextrose. Yield of the enzyme was increased up to 7.53 folds with a specific activity of 55.125 U/mg after gel filtration chromatography. SDS-PAGE analysis confirmed the size of protease as 28.24 kDa. Purified enzyme retained its catalytic activity over a broad range of temperature (5 to 65 °C) and pH [5–8]. Addition of metal ions shown to have a stimulatory effect on catalytic properties while EDTA inhibited the efficiency of the enzyme confirming it as a metalloprotease. Protease exhibited excellent stability and activity in the presence of anionic surfactants, solvents, and detergents. The results of dehairing and destaining experiments suggested that the protease produced by B. subtilis KT004404 could be used in leather and textile industries with ecological benefits.
Keywords: Metalloproteases; Thermotolerance; Bacillus subtilis ; Dehairing; Destaining

Evaluation of Enzymatic Deinking of Non-impact Ink Laser-Printed Paper Using Crude Enzyme from Penicillium rolfsii c3-2(1) IBRL by Kok Chang Lee; Woei Yenn Tong; Darah Ibrahim; Takamitsu Arai; Yoshinori Murata; Yutaka Mori; Akihiko Kosugi (451-463).
Application of microbial enzymes for paper deinking is getting tremendous attention due to the rapidly increasing of waste paper every year. This study reports the deinking efficiency of laser-printed paper by the lignocellulolytic enzyme from Penicillium rolfsii c3-2(1) IBRL strain compared to other enzyme sources as well as commercial available enzymes. High enzymatic deinking efficiency of approximately 82 % on laser-printed paper was obtained by pulp treatment with crude enzyme from P. rolfsii c3-2(1) IBRL. However, this crude enzyme was found to reduce the paper strength properties of the pulp based on the results of tensile, tear and burst indices, most probably due to the cellulose degradation. This was further proven by the low viscosity of paper pulp obtained after enzymatic treatment and increasing of sugar production during the treatment. Balancing to this detrimental effect on paper pulp, high deinking efficiency was achieved within a short period of time, in which the enzymatic treatment was conducted for 30 min that enabled contribution to higher brightness index obtained, thus promoting savings of time and energy consumption, therefore environmental sustainability. Extensive research should be conducted to understand the nature and mechanism of enzymatic deinking process by the crude enzyme from P. rolfsii c3-2(1) IBRL in order to improve paper strength properties.
Keywords: Penicillium rolfsii c3-2(1) IBRL; Enzymatic deinking; Non-impact ink laser-printed paper; Tensile; Tear; Burst; Viscosity

Essential and Beneficial Trace Elements in Plants, and Their Transport in Roots: a Review by Recep Vatansever; Ibrahim Ilker Ozyigit; Ertugrul Filiz (464-482).
The essentiality of 14 mineral elements so far have been reported in plant nutrition. Eight of these elements were known as micronutrients due to their lower concentrations in plants (usually ≤100 mg/kg/dw). However, it is still challenging to mention an exact number of plant micronutrients since some elements have not been strictly proposed yet either as essential or beneficial. Micronutrients participate in very diverse metabolic processes, including from the primary and secondary metabolism to the cell defense, and from the signal transduction to the gene regulation, energy metabolism, and hormone perception. Thus, the attempt to understand the molecular mechanism(s) behind their transport has great importance in terms of basic and applied plant sciences. Moreover, their deficiency or toxicity also caused serious disease symptoms in plants, even plant destruction if not treated, and many people around the world suffer from the plant-based dietary deficiencies or metal toxicities. In this sense, shedding some light on this issue, the 13 mineral elements (Fe, B, Cu, Mn, Mo, Si, Zn, Ni, Cl, Se, Na, Al, and Co), required by plants at trace amounts, has been reviewed with the primary focus on the transport proteins (transporters/channels) in plant roots. So, providing the compiled but extensive information about the structural and functional roles of micronutrient transport genes/proteins in plant roots.
Keywords: Toxicity; Deficiency; Micronutrient; Beneficial element; Broad range affinity