Applied Biochemistry and Biotechnology (v.183, #3)

Release of Polyphenols Is the Major Factor Influencing the Bioconversion of Rice Straw to Lactic Acid by Xingxuan Chen; Yiyun Xue; Jiajun Hu; Yiu Fai Tsang; Min-Tian Gao (685-698).
In this study, we found that p-coumaric acid (p-CA), ferulic acid (FA), and condensed tannins were released from rice straw during saccharification. The presence of polyphenols prolonged the lag phase and lowered the productivity of lactic acid. p-CA was identified as a key inhibitor. Tannins had a lower inhibitory effect than p-CA; FA had little inhibitory effect. Acid, alkaline, and ball milling pretreatments elicited different levels of polyphenol release from rice straw. Due to the different levels of polyphenol release in the pretreatment step, the enzymatic hydrolysates contained different concentrations of polyphenols. Compared with fermentation with a synthetic medium, fermentation with the hydrolysates of ball-milled rice straw provided much lower productivity and yield of lactic acid due to the presence of polyphenols. Removal of these compounds played an important role in lactic acid fermentation. When rice straw was alkaline pretreated, the hydrolysates contained few phenolic compounds, resulting in high productivity and yield of lactic acid (1.8 g/L/h and 26.7 g/100 g straw), which were comparable to those in a synthetic medium. This indicates that there is a correlation between removal of phenolic compounds and efficiency in lactic acid fermentation.
Keywords: Lactic acid; Lactobacillus rhamnosus ; Phenolic compounds; Rice straw

Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from Anabaena variabilis by Modification of Center-Near Surface Residues by Fan Zhang; Nan Huang; Li Zhou; Wenjing Cui; Zhongmei Liu; Longbao Zhu; Yi Liu; Zhemin Zhou (699-711).
Phenylalanine ammonia lyase from Anabaena variabilis (Av-PAL) is a candidate for the treatment of phenylketonuria (PKU). However, Av-PAL shows its optimal pH at 8.5 and maintains only 70% of its highest activity when pH decreases to 7.3–7.4 (the condition of human plasma). The objective of the study was to shift its optimal pH by mutating surface amino acid residues which interact with the general base Tyr78. Based on the crystal structure and the online program GETAREA, we selected five sites: Asn69, Glu72, Glu75, Asn89, and Val90. Removing negative charges or introducing positive charges near the general base Tyr78 by mutation, the pH optima were successfully shifted to more acidic range. Especially, the pH optima of E75A, E75L, and E75Q were shifted to 7.5 with 35, 30, and 24% higher specific activities than that of the wild, respectively. Half-lives of E75L and E75Q at 70 °C prolonged to 190 and 180 min from 130 min of the wild, respectively. In addition, the higher resistance to a low pH of 3.5 and protease made E75L a candidate for oral medicine of PKU. This work would improve the therapeutic prospect of Av-PAL and provide guidance in modulating optimal pH of enzymes.
Keywords: Phenylalanine ammonia lyase; pH shift; Therapeutic application; Mutation; Surface residues; Kinetic stability; Resistance

This study evaluated the production of hydrogen and propionic acid in an expanded granular sludge bed (EGSB) reactor by co-fermentation of cheese whey (CW) and crude glycerol (CG). The reactor was operated at hydraulic retention time (HRT) of 8 h by changing the CW/CG ratio from 5:1 to 5:2, 5:3, 5:4, and 5:5. At the ratio of 5:5, HRT was reduced from 8 to 0.5 h. The maximum hydrogen yield of 0.120 mmol H2 g COD−1 was observed at the CW/CG ratio of 5:1. Increasing the CG concentration repressed hydrogen production in favor of propionic acid, with a maximum yield of 6.19 mmol HPr g COD−1 at the CW/CG ratio of 5:3. Moreover, by reducing HRT of 8 to 0.5 h, the hydrogen production rate was increased to a maximum value of 42.5 mL H2 h−1 L−1at HRT of 0.5 h. The major metabolites were propionate, 1,3-propanediol, acetate, butyrate, and lactate.
Keywords: EGSB; 1,3-Propanediol; Propionic acid; Cheese whey; Crude glycerol

Effects of Feedstock Sources on Inoculant Acclimatization: Start-up Strategies and Reactor Performance by Qian Yang; Liang-Huan Wei; Wei-Zun Li; Yu Chen; Mei-Ting Ju (729-743).
Different inoculum sources and acclimatization methods result in different substrate adaptation and biodegradability. To increase straw degradation rate, shorten the digester start-up time, and enhance the biogas production, we domesticated anaerobic sludge by adding microcrystalline cellulose (MCC). During acclimatization, the start-up strategies and reactor performance were investigated to analyze changes in feedstock adaption, biodegradability, and methanogen activity. The effect of the domesticated inoculum was evaluated by testing batch un-pretreated corn stover with a dewatered sludge (DS)-domesticated inoculum as a control. The results showed that (1) using MCC as a substrate rapidly improved microorganism biodegradability and adaptation. (2) MCC as domesticated substrate has relatively stable system and high mass conversion, but with low buffer capacity. (3) Macro- and micronutrients should be added for improving the activity of methanogenic and system’s buffer capacity. (4) Using the domesticated inoculums and batch tests to anaerobically digest untreated corn stover yielded rapid biogas production of 292 mL, with an early peak value on the first day. The results indicated that cultivating directional inoculum can efficiently and quickly start-up digester. These investigated results to promote anaerobic digestion of straw for producing biogas speed up the transformation of achievements of biomass solid waste utilization have a positive promoting significance.
Keywords: Inoculum; Acclimatization; Microcrystalline cellulose; Dewatered sludge; Anaerobic digestion

Beneficial Effects of Phyllanthus amarus Against High Fructose Diet Induced Insulin Resistance and Hepatic Oxidative Stress in Male Wistar Rats by Mallaiah Putakala; Sudhakara Gujjala; Srinivasulu Nukala; Saralakumari Desireddy (744-764).
Insulin resistance (IR) is a characteristic feature of obesity, type 2 diabetes mellitus, and cardiovascular diseases. Emerging evidence suggests that the high-fructose consumption is a potential and important factor responsible for the rising incidence of IR. The present study investigates the beneficial effects of aqueous extract of Phyllanthus amarus (PAAE) on IR and oxidative stress in high-fructose (HF) fed male Wistar rats. HF diet (66% of fructose) and PAAE (200 mg/kg body weight/day) were given concurrently to the rats for a period of 60 days. Fructose-fed rats showed weight gain, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired insulin sensitivity, dyslipidemia, hyperleptinemia, and hypoadiponectinemia (P < 0.05) after 60 days. Co-administration of PAAE along with HF diet significantly ameliorated all these alterations. Regarding hepatic antioxidant status, higher lipid peroxidation and protein oxidation, lower reduced glutathione levels and lower activities of enzymatic antioxidants, and the histopathological changes like mild to severe distortion of the normal architecture as well as the prominence and widening of the liver sinusoids observed in the HF diet-fed rats were significantly prevented by PAAE treatment. These findings indicate that PAAE is beneficial in improving insulin sensitivity and attenuating metabolic syndrome and hepatic oxidative stress in fructose-fed rats.
Keywords: Insulin resistance; High-fructose diet; Oxidative stress; Oral glucose tolerance test; Phyllanthus amarus

Evaluation of Chitosan-Based Films Containing Gelatin, Chondroitin 4-Sulfate and ZnO for Wound Healing by Thiago B. Cahú; Rodrigo A. Silva; Raquel P. F. Silva; Milena M. Silva; Isabel R. S. Arruda; Janilson F. Silva; Romero M. P. B. Costa; Suzan D. Santos; Helena B. Nader; Ranilson S. Bezerra (765-777).
In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 ± 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.
Keywords: Waste biotechnology; Biopolymer dressing; Biocompatibility; Zinc oxide; Antimicrobial

Preparation of Biodiesel with Liquid Synergetic Lipases from Rapeseed Oil Deodorizer Distillate by Leping Zeng; Yaojia He; Liangcheng Jiao; Kai Li; Yunjun Yan (778-791).
To reduce industrial production cost, cheap and easily available rapeseed oil deodorizer distillates were used as feedstock to prepare biodiesel in this study. As a result, liquid forms of Candida rugosa lipase and Rhizopus oryzae lipase (ROL) were functioned as new and effective catalysts with biodiesel yield of 92.63% for 30 h and 94.36% for 9 h, respectively. Furthermore, the synergetic effect between the two lipases was employed to enhance biodiesel yield with a result of 98.16% in 6 h under optimized conditions via response surface methodology. The obtained conversion rate surpassed both yields of the individual two lipases and markedly shortened the reaction time. The resultant optimal conditions were ROL ratio 0.84, water content 46 wt% (w/w), reaction temperature 34 °C, and reaction time 6 h.
Keywords: Rapeseed oil deodorizer distillates (RODDs); Biodiesel; Liquid lipase; Candida rugosa lipase; Rhizopus oryzae lipase; Synergetic effect

Kinetics of Enzymatic Synthesis of Cinnamyl Butyrate by Immobilized Lipase by Govind V. Waghmare; Abhishek Chatterji; Virendra K. Rathod (792-806).
This work illustrates the enzymatic synthesis of cinnamyl butyrate by esterification of butyric acid and cinnamyl alcohol. Experiments were performed to study the various operating parameters such as molar ratio, enzyme concentration, temperature, and speed of agitation. Also, the suitable kinetic model for esterification reaction was predicted and the various kinetic parameters were determined. It has been observed that the experimental results agree well with the simulated results obtained by following the ping-pong bi-bi mechanism with dead-end inhibition by both the substrate acid and alcohol. The highest 90% conversion of butyric acid was observed after 12 h at the following reaction conditions: substrate molar ratio 1:2 (butyric acid/cinnamyl alcohol), temperature 50 °C, enzyme loading 2% (with respect to the weight of the substrates), and agitation speed 250 rpm. Diffusional mass transfer limitations between substrate and enzyme surface do not show significant effect on reaction kinetics. Enzyme reusability study reveals that it retains 85% of its catalytic activity after five consecutive cycles.
Keywords: Esterification; Flavor synthesis; Cinnamyl butyrate; Enzymatic catalysis; Immobilized lipase; Kinetic study; Ping-pong bi-bi mechanism

Immobilization of Candida antarctic Lipase B on Functionalized Ionic Liquid Modified MWNTs by Xiaomei Wan; Susu Tang; Xinran Xiang; He Huang; Yi Hu (807-819).
Multiwalled carbon nanotubes (MWNTs) were modified by imidazole-based ionic liquids with different alkyl groups. The modified support samples were characterized by scanning transmission electron microscopy, Raman spectra, thermogravimetric analyses, and X-ray photoelectron spectroscopy. The samples were used to immobilize Candida antarctic lipase (CALB) and the influence of alkyl chain length of ionic liquids on enzymatic properties was investigated by the hydrolysis reaction of triacetin. The results revealed that functionalized ionic liquids modification did not destroy the structure of MWNTs. Compared with the immobilized CALB on MWNTs, the immobilized CALB on novel carriers all exhibited higher activity, thermal stability, and reusability. Especially, the activity of MWNTs-IL (8C)-CALB improved 15.23-folds than MWNTs-CALB, meanwhile, after incubation at 70 °C for 20 min, residual enzyme activity of MWNTs-IL (8C)-CALB was 46% of the initial activity, while MWNTs-CALB already lost all activity. Besides, MWNTs-IL (8C)-CALB retained 64.5% of its initial activity after 4 cycles, while MWNTs-CALB retained only 2.12%. Graphical Abstract ᅟ
Keywords: Candida antarctic lipase B; Ionic liquids; Carbon nanotubes; Modification; Enzyme immobilization

Bioconversion of Welan Gum from Kitchen Waste by a Two-Step Enzymatic Hydrolysis Pretreatment by Panyu Li; Yi Xie; Yu Zeng; Wanrong Hu; Yan Kang; Xiang Li; Yabo Wang; Tonghui Xie; Yongkui Zhang (820-832).
Kitchen waste (KW) is a worldwide issue, which can lead to environment pollution. Nevertheless, it is also a low-cost and sustainable resource for bio-production. Meanwhile, welan gum (WG) is one kind of the most important exopolysaccharide but with high material cost. The aim of this study was to adopt two-step enzymatic hydrolysis to improve the release and recovery of both sugar and protein in KW for subsequent WG production. As the results, the recovery rates of sugar and protein reached 81.07 and 77.38%, which were both satisfactory. After the conditions optimized in flasks, the welan fermentation was conducted in a 5-L fermentor, and the WG yield, utilization rates of reducing sugar and KDN, respectively, reached 5.57 g L−1, 94.25% and 61.96%. Moreover, the kinetic analyses demonstrated that the WG fermentation in KWH was a partly growth-associated process. The KW was successfully treated by fermentation for the bioconversion to WG.
Keywords: Kitchen waste; Welan gum; Enzyme catalysis; Biomass conversion; Waste utilization

Carcinogenic Activities and Sperm Abnormalities of Methicillin Resistance Staphylococcus aureus and Inhibition of Their Virulence Potentials by Ayamycin by Mervat Morsy Abbas Ahmed El-Gendy; Khaled G. Abdel-Wahhab; Fathia A. Mannaa; Ayman A. Farghaly; Ahmed M. A. El-Bondkly (833-852).
This investigation aimed to study the in vivo harmful effects of the subcutaneous injection of different methicillin resistance Staphylococcus aureus extracts (MRSA2, MRSA4, MRSA10, MRSA69, MRSA70, MRSA76, and MRSA78). Such strains represented the highest minimum inhibition concentration toward methicillin with various multidrug-resistant patterns. The obtained results revealed that rats injected with the MRSA4 extract died immediately after the last dose indicating the high cytotoxicity of MRSA4 strain (100% mortality). While the mortalities in other groups injected by the other MRSA extracts ranged from 50 to 75%. In comparison with the normal animal group, all MRSA extracts induced a hepatotoxic effect which was indicated from the significant (p < 0.01) increases in the activities of the serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) enzymes. Moreover, alkaline phosphatase (ALP) combined with a partial nephrotoxicity that was monitored from the significant elevation of serum urea concentration. While serum creatinine levels did not affect. Similarly, a significant elevation was recorded in serum levels of tumor biomarkers (alpha fetoprotein; AFP, carcinoembryonic antigen; CEA, and lactate dehydrogenase; LDH) reflecting their carcinogenic potential. On the other hand, the percentage of micronuclei (MN) in polychromatic erythrocytes from bone marrow cells was statistically significant in all groups as compared to the control group. The percentage of sperm abnormalities was statistically significant compared to the control. Different types of head abnormalities and coiled tail were recorded. Consequently, the current study focused on fighting MRSA virulence factors by the new compound ayamycin, which proved to be potent anti-virulence factor against all MRSA strains under study by significant decreasing of their streptokinase activities, hemolysin synthesis, biofilm formation, and their cell surface hydrophobicity.
Keywords: Staphylococcus aureus (MRSA); Carcinogenic activity; Sperm abnormality; Anti-virulent; Streptokinase; Biofilm; Hemolysin

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer’s disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer’s disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.
Keywords: Stable transfection; Amyloid precursor protein; Amyloid-beta; Alzheimer’s disease; Swedish mutation; Swedish/Indiana mutation

Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi by Xinping Lin; Sasa Liu; Ruiqi Bao; Ning Gao; Sufang Zhang; Rongqian Zhu; Zongbao Kent Zhao (867-875).
Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.
Keywords: Lipomyces starkeyi ; Agrobacterium-mediated transformation; Promoters; Genetic engineering; Biofuels

Giant reed, miscanthus, and switchgrass are considered prominent lignocellulosic feedstocks to obtain fermentable sugars for biofuel production. The bioconversion into sugars requires a delignifying pre-treatment step followed by hydrolysis with cellulase and other accessory enzymes like xylanase, especially in the case of alkali pre-treatments, which retain the hemicellulose fraction. Blends richer in accessory enzymes than commercial mix can be obtained growing fungi on feedstock-based substrates, thus ten selected Trichoderma isolates, including the hypercellulolytic strain Trichoderma reesei Rut-C30, were grown on giant reed, miscanthus, or switchgrass-based substrates. The produced enzymes were used to saccharify the corresponding feedstocks, compared to a commercial enzymatic mix (6 FPU/g). Feedstocks were acid (H2SO4 0.2–2%, w/v) or alkali (NaOH 0.02–0.2%, w/v) pre-treated. A microplate-based approach was chosen for most of the experimental steps due to the large number of samples. The highest bioconversion was generally obtained with Trichoderma harzianum Or4/99 enzymes (78, 89, and 94% final sugar yields at 48 h for giant reed, miscanthus, and switchgrass, respectively), with significant increases compared to the commercial mix, especially with alkaline pre-treatments. The differences in bioconversion yields were only partially caused by xylanases (maximum R 2 = 0.5), indicating a role for other accessory enzymes.
Keywords: Arundo donax ; Miscanthus × giganteus ; Panicum virgatum ; Lignocellulose; Cellulase; Xylanase; Acid pre-treatment; Alkaline pre-treatment; Trichoderma ; Reducing sugars

Biodegradation of BTEX Aromatics by a Haloduric Microbial Consortium Enriched from a Sediment of Bohai Sea, China by Yue Deng; Fenglong Yang; Chunping Deng; Jinshui Yang; Jing Jia; Hongli Yuan (893-905).
This study focused on a haloduric BTEX-degrading microbial consortium EC20 enriched from Bohai Sea sediment. EC20 degraded 87% of BTEX at 435 mg L−1 initial concentration (benzene, toluene, ethylbenzene, and xylenes in equal proportions) in the presence of 3.4% NaCl. 16S rRNA gene-based PCR-DGGE profiles revealed that the dominant bacteria in EC20 were Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes at the phylum level, and Pseudomonas, Mesorhizobium, Achromobacter, Stenotrophomonas, and Halomonas at the genus level. PCR detection of genes coding the key enzymes which participated in BTEX degradation pathways showed that the enriched consortium EC20 contained TOL pathway and TOD pathway to initiate biodegradation of BTEX.
Keywords: BTEX; PCR-DGGE; Biodegradation; Bacterial consortium; Metabolic pathway

A Review of the Anaerobic Digestion of Fruit and Vegetable Waste by Chao Ji; Chui-Xue Kong; Zi-Li Mei; Jiang Li (906-922).
Fruit and vegetable waste is an ever-growing global question. Anaerobic digestion techniques have been developed that facilitate turning such waste into possible sources for energy and fertilizer, simultaneously helping to reduce environmental pollution. However, various problems are encountered in applying these techniques. The purpose of this study is to review local and overseas studies, which focus on the use of anaerobic digestion to dispose fruit and vegetable wastes, discuss the acidification problems and solutions in applying anaerobic digestion for fruit and vegetable wastes and investigate the reactor design (comparing single phase with two phase) and the thermal pre-treatment for processing raw wastes. Furthermore, it analyses the dominant microorganisms involved at different stages of digestion and suggests a focus for future studies.
Keywords: Fruit and vegetable waste; Anaerobic digestion; Co-digestion; Two-phase reactor; Biogas production potential

A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation by Song Li; Xupeng Cao; Yan Wang; Zhen Zhu; Haowei Zhang; Song Xue; Jing Tian (923-930).
With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.
Keywords: FASP; mFASP; Chlamydomonas reinhardtii ; Proteomics; Sample preparation

Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.
Keywords: Lignocellulolytic bacteria; Lignocellulose degradation; Environmental samples; Bacterial isolation; Isolation medium; Taxonomic analysis

Pseudostem of the Musa cavendishii banana plant was submitted to chemical pretreatments with acid (H2SO4 2%, 120 °C, 15 min) and with alkali (NaOH 3%, 120 °C, 15 min), saccharified by commercial enzymes Novozymes® (Cellic CTec2 and HTec2). The influences of the pretreatments on the degradation of the lignin, cellulose and hemicellulose, porosity of the surface, particle crystallinity, and yield in reducing sugars after saccharification (Y RS), were established. Different concentrations of biomass (70 and 100 g/L in dry matter (dm)), with different physical differences (dry granulated, crushed wet bagasse, and whole pseudostem), were used. The broth with the highest Y RS among the different strategies tested was evaporated until the concentration of reducing sugars (RS) was to the order of 100 g/L and fermented, with and without prior detoxification with active carbon. Fermentation was carried out in Erlenmeyer flasks, at 30 °C, initial pH 5.0, and 120 rpm. In comparison to the biomass without chemical pretreatment and to the biomass pretreated with NaOH, the acid pretreatment of 70 g/L of dry granulated biomass enabled greater digestion of hemicellulose, lower index of cellulose crystallinity, and higher Y RS (45.8 ± 0.7%). The RS increase in fermentation broth to 100 g/L, with posterior detoxification, presented higher productivity ethanol (Q P = 1.44 ± 0.02 g/L/h) with ethanol yield (Y P/RS) of 0.41 ± 0.02 g/g. The value of Q P was to the order of 75% higher than Q P obtained with the same broth without prior detoxification.
Keywords: Biofuel; Biomass; Renewable energy; Alternative energy; Ethanol 2G

Bioethanol Production Using Waste Seaweed Obtained from Gwangalli Beach, Busan, Korea by Co-culture of Yeasts with Adaptive Evolution by In Yung Sunwoo; Jung Eun Kwon; Trung Hau Nguyen; Chae Hun Ra; Gwi-Taek Jeong; Sung-Koo Kim (966-979).
Conditions for ethanol production were evaluated using waste seaweed obtained from Gwangalli beach, Busan, Korea, after strong winds on January 15, 2015. Eleven types of seaweed were identified, and the proportions of red, brown, and green seaweed wastes were 26, 46, and 28%, respectively. Optimal pretreatment conditions were determined as 8% slurry content, 286 mM H2SO4 for 90 min at 121 °C. Enzymatic saccharification with 16 units/mL Celluclast 1.5L and Viscozyme L mixture at 45 °C for 48 h was carried out as optimal condition. A maximum monosaccharide concentration of 30.2 g/L was obtained and used to produce ethanol. Fermentation was performed with single or mixed yeasts of non-adapted and adapted Saccharomyces cerevisiae KCTC 1126 and Pichia angophorae KCTC 17574 to galactose and mannitol, respectively. The maximum ethanol concentration and yield of 13.5 g/L and YEtOH of 0.45 were obtained using co-culture of adapted S. cerevisiae and P. angophorae.
Keywords: Waste seaweed; RSM (response surface methodology); Adaptive evolution; Co-culture; Ethanol production

Morphological, Histobiochemical and Molecular Characterisation of Low Lignin Phloem Fibre (llpf) Mutant of Dark Jute (Corchorus olitorius L.) by S. B. Choudhary; I. Chowdhury; R. K. Singh; S. P. Pandey; H. K. Sharma; A. Anil Kumar; P. G. Karmakar; N. Kumari; J. Souframanien; S. J. Jambhulkar (980-992).
Lignin is a versatile plant metabolite challenging high-end industrial applications of several plant products including jute. Application of developmental mutant in regulation of lignification in jute may open up door for much awaited jute based diversified products. In the present study, a novel dark jute (Corchorus olitorius L.) mutant with low lignin (7.23%) in phloem fibre being compared to wild-type JRO 204 (13.7%) was identified and characterised. Unique morphological features including undulated stem, petiole and leaf vein distinguished the mutant in gamma ray irradiated mutant population. Histological and biochemical analysis revealed reduced lignification of phloem fibre cells of the plant. RT-PCR analysis demonstrated temporal transcriptional regulation of CCoAMT1 gene in the mutant. The mutant was found an extremely useful model to study phloem fibre developmental biology in the crop besides acting as a donor genetic stock for low lignin containing jute fibre in dark jute improvement programme.
Keywords: Dark jute; Corchorus olitorius ; Mutant; Lignin; Phloem fibre; CCoAMT1

Curcumin Encapsulated in Milk Exosomes Resists Human Digestion and Possesses Enhanced Intestinal Permeability in Vitro by Monika Vashisht; Payal Rani; Suneel Kumar Onteru; Dheer Singh (993-1007).
Exosomes, the extracellular secretary nano-vesicles, act as carriers of biomolecules to the target cells. They exhibit several attributes of an efficient drug delivery system. Curcumin, despite having numerous bioactive and therapeutic properties, has limited pharmaceutical use due to its poor water solubility, stability, and low systemic bioavailability. Hence, this study aims to enhance the therapeutic potential of curcumin, a model hydrophobic drug, by its encapsulation into milk exosomes. In the present study, we investigated the stability of free curcumin and exosomal curcumin in PBS and in vitro digestive processes. Additionally, their uptake and trans-epithelial transport were studied on Caco-2 cells. Curcumin in milk exosomes had higher stability in PBS, sustained harsh digestive processes, and crossed the intestinal barrier than free curcumin. In conclusion, the encapsulation of curcumin into the exosomes enhances its stability, solubility, and bioavailability. Therefore, the present study demonstrated that milk exosomes act as stable oral drug delivery vehicles.
Keywords: Solubility; Stability; Bioavailability; Caco-2 cells; Oral drug delivery

New Insight into Sugarcane Industry Waste Utilization (Press Mud) for Cleaner Biobutanol Production by Using C. acetobutylicum NRRL B-527 by Pranhita R. Nimbalkar; Manisha A. Khedkar; Shashank G. Gaikwad; Prakash V. Chavan; Sandip B. Bankar (1008-1025).
In the present study, press mud, a sugar industry waste, was explored for biobutanol production to strengthen agricultural economy. The fermentative production of biobutanol was investigated via series of steps, viz. characterization, drying, acid hydrolysis, detoxification, and fermentation. Press mud contains an adequate amount of cellulose (22.3%) and hemicellulose (21.67%) on dry basis, and hence, it can be utilized for further acetone-butanol-ethanol (ABE) production. Drying experiments were conducted in the temperature range of 60–120 °C to circumvent microbial spoilage and enhance storability of press mud. Furthermore, acidic pretreatment variables, viz. sulfuric acid concentration, solid to liquid ratio, and time, were optimized using response surface methodology. The corresponding values were found to be 1.5% (v/v), 1:5 g/mL, and 15 min, respectively. In addition, detoxification studies were also conducted using activated charcoal, which removed almost 93–97% phenolics and around 98% furans, which are toxic to microorganisms during fermentation. Finally, the batch fermentation of detoxified press mud slurry (the sample dried at 100 °C and pretreated) using Clostridium acetobutylicum NRRL B-527 resulted in a higher butanol production of 4.43 g/L with a total ABE of 6.69 g/L.
Keywords: Biobutanol; Detoxification; Drying; Fermentation; Press mud; Pretreatment

Microbial Catalyzed Regio-Selective Demethylation of Colchicine by Streptomyces griseus ATCC 13273 by Chao Zhang; Xian Sun; Shao Hua Xu; Bo Yang Yu; Jian Zhang (1026-1034).
Colchicinoids and their derivatives are of great importance in pharmaceutical applications, and colchicine is usually used as the first choice for the treatment of gout. To expand the structural diversities and clinical application of colchicinoids, many attempts have been established for the derivatives with better activity or less toxicity. Herein, in this paper, we report a direct microbial transformation of colchicine into 2-O-demethyl-colchicine (M1) and 3-O-demethl-colchicine (M2) by Streptomyces griseus ATCC 13273. It is noteworthy that when DMF was used as co-solvent, the yield of M1 and M2 could reach up to 51 and 31%, respectively. All the structures of the metabolites were elucidated unambiguously by ESI-MS, 1H–NMR, 13C–NMR, and 2D–NMR spectroscopy.
Keywords: Colchicine; Streptomyces griseus ATCC 13273; Biotransformation; Microbial demethylation; Conditions optimization

Protocatechuate 3,4-dioxygenase (P34O), which is isolated from Rhizobium sp. LMB-1, catalyzes the ring cleavage step in the metabolism of aromatic compounds, and has great potential for environmental bioremediation. However, its structure is very sensitive to different environmental factors, which weaken its activity. Immobilization of the enzyme can improve its stability, allow reusability, and reduce operation costs. In this work, the relative molecular mass of the native P34O enzyme was determined to be 500 kDa by gel filtration chromatography on Sephadex G-200, and the enzyme was immobilized onto (3-aminopropyl) triethoxysilane-modified Fe3O4 nanoparticles (NPs) by the glutaraldehyde method. The optimum pH of immobilized and free P34O was unaffected, but the optimum temperature of immobilized P34O increased from 60 to 70 °C, and the thermal stability of immobilized P34O was better than that of the free enzyme and showed higher enzymatic activity at 60 and 70 °C. In addition, with the exception of Fe3+, most metal ions and organic chemicals could not improve the activity of free and immobilized P34O. The kinetic parameters of the immobilized P34O were higher than those of the free enzyme, and immobilized P34O on Fe3O4 NPs could be reused ten times without a remarkable decrease in enzymatic activity.
Keywords: Protocatechuate 3,4-dioxygenase; Immobilization; Fe3O4 nanoparticles; Thermal stability; Recycling

Ampicillin Mineralization by Denitrifying Process: Kinetic and Metabolic Effects by Irasema Islas-García; Claudia Romo-Gómez; Flor de María Cuervo-López (1049-1061).
The impact of the antibiotic ampicillin (AMP) on the metabolic and kinetics of denitrification process as well as the sludge ability for oxidizing it was evaluated in batch assays. Denitrifying reference assays with acetate-C and nitrate-N (C/N ratio of 1.1) were conducted for establishing the metabolic and kinetic performance of the denitrifying sludge. Assays amended with 10 mg AMP-C L−1 were also performed. In reference assays, acetate and nitrate consumption efficiencies of 100% with a total conversion to HCO3 and N2 were achieved within 1.5 h. When 10 mg AMP-C L−1 was added, total and simultaneous consumption of nitrate-N, acetate-C, and AMP-C was achieved within 12 h. The substrates were completely reduced to N2 and oxidized to HCO3 , respectively. No nitrite-N was registered at the end of culture. AMP caused a reversible inhibitory effect on specific nitrate and acetate consumption and N2 production rates. Complete consumption and mineralization of AMP associated to nitrate reduction to N2 were achieved. This work provides the first evidences on the metabolic and kinetic performance of a denitrifying sludge exposed to AMP. These results might be considered for proposing useful wastewater treatments where β-Lactam antibiotics can be present.
Keywords: Ampicillin (AMP); β-Lactam antibiotic; Denitrification

Co-culture of microalgae with many types of bacteria usually comes out with significant different treatment efficiencies for COD, nitrogen, and phosphorus in wastewater remediation, compared with the single culture. In order to understand the mechanism behind, a comparative experiment was designed in this study, using the green microalgae species Chlorella sorokiniana in the single culture and a consortium with a bacterium, Pseudomonas H4, for nutrient removal. Comparative metabolome profile analysis was conducted to reveal the Chlorella cell responses to the synergistic growth with the bacteria, and possible relations between the metabolic regulation of microalgae and the nutrient degradation were discussed. The detectable differential metabolites of Chlorella belonged to several classes, including carbohydrates, fatty acids, amino acids, phosphates, polyols, etc. The orthogonal partial least squares discriminant analysis (OPLS-DA) model of the identified metabolites suggests the metabolism in this alga was significantly affected by the bacteria, corresponding to different treatment behaviors.
Keywords: Comparative metabolomics; Microalgae; Chlorella ; Consortium; Wastewater remediation

Performance of microbial carbon capture cells (MCCs), having a low-cost clayware separator, was evaluated in terms of wastewater treatment and electricity generation using algae Chlorella pyrenoidosa in MCC-1 and Anabaena ambigua in MCC-2 and without algae in a cathodic chamber of MCC-3. Higher power production was achieved in MCC-1 (6.4 W/m3) compared to MCC-2 (4.29 W/m3) and MCC-3 (3.29 W/m3). Higher coulombic efficiency (15.23 ± 1.30%) and biomass production (66.4 ± 4.7 mg/(L*day)) in MCC-1 indicated the superiority of Chlorella over Anabaena algae for carbon capture and oxygen production to facilitate the cathodic reduction. Algal biofilm formation on the cathode surface of MCC-1 increased dissolved oxygen in the catholyte and decreased the cathodic charge transfer resistance with increase in reduction current. Electrochemical analyses revealed slow cathodic reactions and increase in internal resistance in MCC-2 (55 Ω) than MCC-1 (30 Ω), due to lower oxygen produced by Anabaena algae. Thus, biomass production in conjunction with wastewater treatment, CO2 sequestration and electricity generation can be achieved using Chlorella algal biocathode in MCC.
Keywords: Chlorella algae; Electrochemical analyses; Microbial carbon capture cell; Microbial fuel cell; Wastewater treatment

Evaluation of Brown Midrib Sorghum Mutants as a Potential Biomass Feedstock for 2,3-Butanediol Biosynthesis by Yadhu N Guragain; P Srinivasa Rao; PV Vara Prasad; Praveen V Vadlani (1093-1110).
Three sorghum backgrounds [Atlas, Early Hegari (EH), and Kansas Collier (KC)] and two bmr mutants (bmr6 and bmr12) of each line were evaluated and compared for grain and biomass yield, biomass composition, and 2,3-butanediol production from biomass. The data showed that the bmr6 mutation in EH background led to a significant decrease in stover yield and increase in grain yield, whereas the stover yield was increased by 64% without affecting grain yield in KC background. The bmr mutants had 10 to 25% and 2 to 9% less lignin and structural carbohydrate contents, respectively, and 24 to 93% more non-structural sugars than their parents in all sorghum lines, except EH bmr12. The total fermentable sugars released were 22 to 36% more in bmr mutants than in parents for Atlas and KC, but not for EH. The bmr6 mutation in KC background produced the most promising feedstock, among the evaluated bmr mutants, for 2,3-butanediol production without affecting grain yield, followed by KC bmr12 and Atlas bmr6, but the bmr mutation had an adverse effect in EH background. This indicated that the genetic background of the parent line and type of bmr mutation significantly affect the biomass quality as a feedstock for biochemical production.
Keywords: Bmr ; Biomass composition; Pretreatment; Fermentation; 2,3-Butanediol; Platform chemical; Stover (dry biomass)

Two kinds of exoglucanase were purified from a marine Aspergillus niger. Catalytic ability of halophilic exoglucanase with a lower molecular weight and secondary structure change was analyzed at different salinities. Activity of the low molecular weight exoglucanase in 10% NaCl solution (w/v) was 1.69-fold higher of that in NaCl-free solution. Half-life time in 10% NaCl solution (w/v) was over 1.27-fold longer of that in NaCl-free solution. Free energy change of the low molecular weight exoglucanase denaturation, △G, in 10% NaCl solution (w/v) was 0.54 kJ/mol more than that in NaCl-free solution. Melt point in 10% NaCl solution (w/v), 52.01 °C, was 4.21 °C higher than that in NaCl-free solution, 47.80 °C. K m value, 0.179 mg/ml in 10% NaCl solution (w/v) was less 0.044 mg/ml than that, 0.224 mg/ml, in NaCl-free solution. High salinity made content of α-helix increased. Secondary structure change caused by high salinities improved exoglucanase thermostability and catalysis activity. The halophilic exoglucanase from a marine A. niger was valuable for hydrolyzing cellulose at high salinities.
Keywords: Halophilic exoglucanase; Thermodynamic kinetics; High salinity; Marine Aspergillus niger