Applied Biochemistry and Biotechnology (v.167, #6)

Bioethanol Production Involving Recombinant C. thermocellum Hydrolytic Hemicellulase and Fermentative Microbes by Saprativ P. Das; Rajeev Ravindran; Shadab Ahmed; Debasish Das; Dinesh Goyal; Carlos M. G. A. Fontes; Arun Goyal (1475-1488).
The enhancement of the biomass productivity of Escherichia coli cells harbouring the truncated 903 bp gene designated as glycoside hydrolase family 43 (GH43) from Clostridium thermocellum showing hemicellulase activity along with its further use in simultaneous saccharification and fermentation (SSF) process is described. (Phosphoric acid) H3PO4–acetone treatment and ammonia fibre expansion (AFEX) were the pretreatment strategies employed on the leafy biomass of mango, poplar, neem and asoka among various substrates owing to their high hemicellulose content. GH43 showed optimal activity at a temperature of 50 °C, pH 5.4 with stability over a pH range of 5.0–6.2. A 4-fold escalation in growth of the recombinant E. coli cells was observed when grown using repeated batch strategy in LB medium supplemented with glucose as co-substrate. Candida shehatae utilizing pentose sugars was employed for bioethanol production. AFEX pretreatment proved to be better over acid–acetone technique. The maximum ethanol concentration (1.44 g/L) was achieved for AFEX pretreated mango (1%, w/v) followed by poplar with an ethanol titre (1.32 g/L) in shake flask experiments. A 1.5-fold increase in ethanol titre (2.11 g/L) was achieved with mango (1%, w/v) in a SSF process using a table top 2-L bioreactor with 1 L working volume.
Keywords: Simultaneous saccharification and fermentation (SSF); Repeated batch; Glycoside hydrolase family 43(GH43); Clostridium thermocellum ; H3PO4–acetone; AFEX; Candida shehatae

An Evaluation of Chemical Pretreatment Methods for Improving Enzymatic Saccharification of Chili Postharvest Residue by Varghese Elizabeth Preeti; Soolamkandath Variem Sandhya; Mathiyazhakan Kuttiraja; Raveendran Sindhu; Sankar Vani; Sukumaran Rajeev Kumar; Ashok Pandey; Parameswaran Binod (1489-1500).
Residue of chili plants left in the field after harvesting is a major lignocellulosic resource that is underexploited. India has over 0.6 million tons of this residue available as surplus annually which projects it as a potent feedstock for conversion to bioethanol. The cellulose, hemicellulose and lignin content of the chili residues are subject to variations with type of cultivar, geographical region and the season of cultivation, and the composition is critical in developing strategies for its conversion to bioalcohol(s). As with any lignocellulosic biomass, this feedstock needs pretreatment to make it more susceptible to hydrolysis by enzymes which is the most efficient method for generating sugars which can, then, be fermented to alcohol. Pretreatment of chili postharvest residue (CPHR) is, therefore, important though very little study has addressed this challenge. Similarly, enzymatic saccharification of pretreated chili biomass is another area which needs dedicated R&D because the combination of enzyme preparations and the conditions for saccharification are different in different biomass types. The present study was undertaken to develop an optimal process for pretreatment and enzymatic saccharification of CPHR that will yield high amount of free sugars. Dilute acid and alkali pretreatment of the biomass was studied at high temperatures (120–180 °C), with mixing (50–200 rpm) in a high pressure reactor. The holding time was adjusted between 15 and 60 min, and the resultant biomass was evaluated for its susceptibility to enzymatic hydrolysis. Similarly, the conditions for hydrolysis including biomass and enzyme loadings, mixing and incubation time were studied using a Taguchi method of experimentation and were optimized to obtain maximal yield of sugars. Efficiency of pretreatment was gauged by observing the changes in composition and the physicochemical properties of native and pretreated biomass which were analyzed by SEM and XRD analyses. The studies are expected to provide insights into the intricacies of biomass conversion leading to better processes that are simpler and more efficient.
Keywords: Chili postharvest residue; Bioethanol; Pretreatment; Cellulase; Biomass hydrolysis; Biomass composition

Isolation and Characterization of Zygomycetes Fungi from Tempe for Ethanol Production and Biomass Applications by Rachma Wikandari; Ria Millati; Patrik R. Lennartsson; Eni Harmayani; Mohammad J. Taherzadeh (1501-1512).
Mixed fungal cultures used for making tempe, a fermented soy bean food, were screened for biomass conversion. Thirty-two zygomycetes strains from two tempe cultures were isolated and identified as Rhizopus, Mucor, Rhizomucor, and Absidia species based upon morphology. The dry weight biomass of these strains contained 49% to 63% protein and 10–24% chitosan. The strains with the best growth performance were selected and registered at Culture Collection of Gothenburg University as Rhizomucor CCUG 61146 and Rhizomucor CCUG 61147. These strains were able to grow both aerobically and micro-aerobically. Their ethanol yields were 0.38–0.47, 0.19–0.22, and 0.31–0.38 g/g on glucose, xylose, and a mix sugars consisting of cellobiose, glucose, xylose, arabinose, galactose, and mannose, respectively. The biomass yield of the strains varied between 65 and 140 mg dry weight/g glucose.
Keywords: Ethanol; Zygomycetes; Tempe; Chitosan

Surfactant-Assisted Acid Pretreatment of Sugarcane Tops for Bioethanol Production by Raveendran Sindhu; Mathiyazhakan Kuttiraja; Parameswaran Binod; Varghese Elizabeth Preeti; Soolankandath Variem Sandhya; Sankar Vani; Rajeev Kumar Sukumaran; Ashok Pandey (1513-1526).
Sugarcane tops is one of the largest biomass resources in India and in tropical countries such as Brazil in terms of surplus availability. Conversion of this feedstock to ethanol requires pretreatment to make it more accessible for the enzymes used in saccharification. Though several pretreatment regimens have been developed for addressing biomass recalcitrance, very few seem to be promising as an industrial process. A novel hybrid method involving use of mild acid and surfactant was developed which could effectively remove lignin and improve the sugar yield from sugar cane tops. Operational parameters that affect the pretreatment efficiency (measured as yield of sugars) were studied and optimized. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method could yield 0.798 g of reducing sugars per gram of pretreated biomass upon enzymatic hydrolysis.
Keywords: Sugarcane tops; Bioethanol; Pretreatment; Saccharification; Surfactant

Kinetic Study of Empty Fruit Bunch Using Hot Liquid Water and Dilute Acid by Jun Seok Kim; Won Il Choi; Minsu Kang; Ji Yeon Park; Jin-Suk Lee (1527-1539).
Empty fruit bunch (EFB), a residual product of the palm plantation, is an attractive biomass for biorefinery. As xylan is susceptible to high temperature pretreatment, it is important to setup a proper pretreatment condition to maximize the sugar recovery from EFB. Kinetic parameters of mathematical models were obtained in order to predict the concentration of xylose, glucose, furfural, and acetic acid in the hydrolysate and to find production conditions of xylose. We investigated the kinetics of hot liquid water and dilute sulfuric acid hydrolysis over a 40-min period using a self-designed setup by measuring the concentrations of released sugars (xylose, glucose) and degradation products (acetic acid and furfural). The reaction was performed within the range 160∼180 °C, under reaction conditions of various concentration of sulfuric acid (0.1∼0.2%) and 1:7 solid–liquid ratio in a batch reactor. The kinetic constants can be expressed by the Arrhenius equation with the activation energy for the hydrolysis of sugar and decomposition of sugar. The activation energy of xylose was determined to be 136.2187 kJ mol−1.
Keywords: Empty bunch fruit (EFB); Kinetics; Xylan; Acid hydrolysis; Hot liquid water (HLW)

Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process by Pankaj K. Rai; S. P. Singh; R. K. Asthana (1540-1549).
Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (∼79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H2 yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H2 yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H2 yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H2 yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H2 yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H2 production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H2 production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.
Keywords: Enterobacter ; Rhodopseudomonas ; Whey; H2 production

The Assessment of Phytoremediation Potential of Invasive Weed Amaranthus spinosus L. by M. Devi Chinmayee; B. Mahesh; S. Pradesh; I. Mini; T. S. Swapna (1550-1559).
Presently the environment is heavily polluted by various toxic metals, which creates danger for all living beings. Heavy metals are toxic above certain threshold levels. Phytoremediation is an emerging technology which is quite a novel technique of cleaning polluted sites through the use of plants. Phytoremediation methods are comparatively cheap and ecologically advantageous, compared to conventional and physicochemical methods like precipitation, evaporation and chemical reduction. In this respect, plants can be compared to solar-driven pumps capable of extracting and concentrating certain elements from their environment. Amaranthus spinosus, an invasive weed seen on road sides and bare land belonging to the family Amaranthaceae, was selected for the present study. A greenhouse experiment was conducted and consisted of a range-finding test and definitive test for various concentrations of heavy metals Cu, Zn, Cr, Pb and Cd. Plants were grown in soil treated with different concentration of metals depending upon the threshold level. The bio-organics of the plant such as soluble sugar, protein, lipid, phenol, amino acid and photosynthetic pigments were estimated after 30 days of treatment. The bio-organics showed profound variation in response to accumulation of heavy metals. Accumulation of Cu, Pb and Cd was high in the roots followed by stem and leaves and that of Zn and Cr remained high in aerial parts. A steady increase was noticed in the bioaccumulation of copper, zinc and cadmium on enhancing the concentration of the corresponding metal in the soil. The bioconcentration factor and translocation factor were above unity in most of the treatments and increased as the concentration of treatment increased which indicated that A. spinosus is a potential agent for heavy metal accumulation and translocation.
Keywords: Phytoremediation; Amaranthus spinosus ; Amaranthaceae; Heavy metals; BCF; TF

The significant challenges presented by the April 20, 2010 explosion, sinking, and subsequent oil spill of the Deepwater Horizon drilling platform in Canyon Block 252 about 52 miles southeast of Venice, LA, USA greatly impacted Louisiana’s coastal ecosystem including the sea food industry, recreational fishing, and tourism. The short-term and long-term impact of this oil spill are significant, and the Deepwater Horizon spill is potentially both an economic and an ecological disaster. Microbes present in the water column and sediments have the potential to degrade the oil. Oil degradation could be enhanced by biostimulation method. The conventional approach to bioremediation of petroleum hydrocarbon is based on aerobic processes. Anaerobic bioremediation has been tested only in a very few cases and is still considered experimental. The currently practiced conventional in situ biorestoration of petroleum-contaminated soils and ground water relies on the supply of oxygen to the subsurface to enhance natural aerobic processes to remediate the contaminants. However, anaerobic microbial processes can be significant in oxygen-depleted subsurface environments and sediments that are contaminated with petroleum-based compounds such as oil-impacted marshes in Louisiana. The goal of this work was to identify the right conditions for the indigenous anaerobic bacteria present in the contaminated sites to enhance degradation of petroleum hydrocarbons. We evaluated the ability of microorganisms under a variety of electron acceptor conditions to degrade petroleum hydrocarbons. Researched microbial systems include sulfate-, nitrate-reducing bacteria, and fermenting bacteria. The results indicated that anaerobic bacteria are viable candidates for bioremediation. Enhanced biodegradation was attained under mixed electron acceptor conditions, where various electron-accepting anaerobes coexisted and aided in degrading complex petroleum hydrocarbon components of marsh sediments in the coastal Louisiana. Significant degradation of oil also occurred under sulfate-reducing and nitrate-reducing conditions.
Keywords: Oil spill; Petroleum hydrocarbon; Anaerobic degradation; Electron acceptors; Food chain

p-Nitrophenol Biodegradation by Aerobic Microbial Granules by E. Suja; Y. Venkata Nancharaiah; Vayalam P. Venugopalan (1569-1577).
Mixed microbial consortia in the form of aerobic microbial granules (AMG) capable of xenobiotic degradation can be developed from activated sludge or by adaptation of microbial granules pre-grown on labile carbon sources. Both of these approaches were investigated for the cultivation of AMG capable of p-nitrophenol (PNP) biodegradation. Attempts to cultivate AMG from activated sludge using PNP as the sole carbon source were not successful due to poor microbial growth and washout of the inoculated activated sludge. As part of the second approach, parallel sequencing batch reactors (SBRs) were inoculated with pre-grown AMG and operated by feeding both acetate and PNP together (RA), PNP alone (RB) or acetate alone (RC). Acetate/PNP mineralization and nitrification were monitored in the three SBRs. PNP biodegradation was quickly established in both RA and RB. PNP removal rates were found to be 47 and 55 mg g VSS−1 h−1 in RA and RB, respectively. PNP biodegradation during the SBR cycle consisted of distinct lag, exponential and deceleration phases. However, with higher concentrations of PNP (>50 mg l−1), disintegration of granules was observed in RA and RB. When PNP was the sole carbon source, it inhibited the development of aerobic granules from activated sludge and caused disintegration of pre-cultivated aerobic granules. When PNP was the co-substrate along with acetate, the structural and functional integrity (including nitrification) of the granular sludge was maintained. This report highlights the importance of a labile co-substrate for maintaining the physical and functional integrity of granular sludge, when used for toxic waste degradation.
Keywords: Aerobic microbial granules; aerobic granular sludge; biodegradation; p-Nitrophenol; Sequencing batch reactor

Decolorization and Biodegradation of Rubine GFL by Microbial Consortium GG-BL in Sequential Aerobic/Microaerophilic Process by Tatoba R. Waghmode; Mayur B. Kurade; Harshad S. Lade; Sanjay P. Govindwar (1578-1594).
This study represents the development of a new batch method by consortium GG-BL using two microbial cultures viz., Galactomyces geotrichum MTCC 1360 and Brevibacillus laterosporus MTCC 2298, by varying environmental conditions for the decolorization and biodegradation of Rubine GFL. Consortium was found to give better decolorization and degradation of Rubine GFL as compared to the individual microorganism at aerobic/microaerophilic process. The consortial metabolic activity of these strains lead to 100% decolorization of Rubine GFL (50 mg/L) within 30 h with significant reduction in chemical oxygen demand (79%) and total organic carbon (68%). Induction in the activities of laccase, veratryl alcohol oxidase, tyrosinase, azo reductase, and riboflavin reductase suggested their role in the decolorization process. Nondenaturing polyacrylamide gel electrophoresis analysis showed differential induction pattern of oxidoreductive enzymes during decolorization of the dye at different incubation temperatures. The degradation of Rubine GFL into different metabolites by individual organism and in consortium was confirmed using high performance thin layer chromatography, high performance liquid chromatography, Fourier transform infrared spectroscopy, and gas chromatography-mass spectroscopy analysis. Phytotoxicity studies revealed nontoxic nature of the metabolites of Rubine GFL.
Keywords: Consortium GG-BL; Rubine GFL; Decolorization; Veratryl alcohol oxidase; PAGE

The present study deals with the isolation of fungi from soil with the ability to degrade polyurethane (PU). A pure fungal isolate was analyzed for its ability to utilize PU as a sole carbon source in shaking culture for 30 days. Incubation of PU with Aspergillus flavus resulted in 60.6% reduction in weight of PU. The scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR) results showed certain changes on the surface of PU film and formation of some new intermediate products after polymer breakdown. Thermogravimetric curves showed changes between the thermal behavior of the samples that were inoculated with A. flavus and control. FTIR spectra showed detectable changes in control and incubated samples, suggesting that degradation occurs, with the decreased intensity of band at 1,715 cm−1, corresponding to ester linkages. We have identified an extracellular esterase activity which might be responsible for the polyurethanolytic activity.
Keywords: Aspergillus ; Polyurethane; Biodegradation; Esterase

Degradation of Triclosan under Aerobic, Anoxic, and Anaerobic Conditions by Prajeesh Gangadharan Puthiya Veetil; Anupama Vijaya Nadaraja; Arya Bhasi; Sudheer Khan; Krishnakumar Bhaskaran (1603-1612).
Triclosan (2, 4, 4′-trichloro-2′-hydroxyl diphenyl ether) is a broad-spectrum antimicrobial agent present in a number of house hold consumables. Aerobic and anaerobic enrichment cultures tolerating triclosan were developed and 77 bacterial strains tolerating triclosan at different levels were isolated from different inoculum sources. Biodegradation of triclosan under aerobic, anoxic (denitrifying and sulphate reducing conditions), and anaerobic conditions was studied in batch cultures with isolated pure strains and enrichment consortium developed. Under aerobic conditions, the isolated strains tolerated triclosan up to 1 g/L and degraded the compound in inorganic-mineral-broth and agar media. At 10 mg/L level triclosan, 95 ± 1.2% was degraded in 5 days, producing phenol, catechol and 2, 4-dichlorophenol as the degradation products. The strains were able to metabolize triclosan and its degradation products in the presence of monooxygenase inhibitor 1-pentyne. Under anoxic/anaerobic conditions highest degradation (87%) was observed in methanogenic system with acetate as co-substrate and phenol, catechol, and 2, 4-dichlorophenol were among the products. Three of the isolated strains tolerating 1 g/L triclosan were identified as Pseudomonas sp. (BDC 1, 2, and 3).
Keywords: Antimicrobial compound; Biodegradation; Pseudomonas sp.; Triclosan

In the present work, evaluation of different conventional techniques, i.e., chemical oxygen demand (COD), biological oxygen demand, elemental analysis, Fourier transform infrared (FTIR), and gas chromatography used for estimating biodegradation of grease waste was carried out. In this order grease waste was incubated with Penicillium chrysogenum for 7–30 days and analyzed percentage of degradation. After 15 days of incubation, the percentages of reduction in COD, carbon, hydrogen, and nitrogen content of grease waste were found 28, 53.5, 12.7, and 0, respectively. Further it was analyzed by FTIR and gas chromatography–mass spectroscopy (GC-MS) and observed that bifurcated peaks of grease waste at 2,926 and 2,855 cm−1 had reduced remarkably, which corresponds to aliphatic hydrocarbons, while new broad peaks appeared at 3,400 cm−1 indicating addition of oxygen molecule to reduced aliphatic hydrocarbon. GC-MS study also supports the results of FTIR, COD, and elemental analysis, but quantification of the percentage of degradation was difficult and limited to volatile organic content, while COD and elemental analysis were found more accurate and more informative. The current study would be helpful in the estimation of biodegradability not only of grease waste but also of other complex nonbiodegradable compounds polluting the environment.
Keywords: Grease waste; Biodegradation; FTIR; GC-MS; COD; BOD; Penicillium chrysogenum

Aerobic Granulation: Advances and Challenges by Kuan-Yeow Show; Duu-Jong Lee; Joo-Hwa Tay (1622-1640).
Aerobic granulation was developed in overcoming the problem of biomass washout often encountered in activated sludge processes. The novel approach to developing fluffy biosolids into dense and compact granules offers a new dimension for wastewater treatment. Compared with conventional biological flocs, aerobic granules are characterized by well-defined shape and compact buildup, superior biomass retention, enhanced microbial functions, and resilient to toxicity and shock loading. This review provides an up-to-date account on development in aerobic granulation and its applications. Granule characterization, factors affecting granulation, and response of granules to various environmental and operating conditions are discussed. Maintaining granule of adequate structural stability is one of the main challenges for practical applications of aerobic granulation. This paper also reviews recent advances in addressing granule stability and storage for use as inoculums, and as biomass supplement to enhance treatment efficiency. Challenges and future work of aerobic granulation are also outlined.
Keywords: Aerobic granulation; Stability; Formation; Review

Bacterial Reduction of Cr(VI) at Technical Scale—The Malaysian Experience by Zainul Akmar Zakaria; Wan Azlina Ahmad; Zainoha Zakaria; Firdausi Razali; Norsuhada Abdul Karim; Mohamad Md Sum; Mohd. Saufi Mohd. Sidek (1641-1652).
The bacterial reduction of Cr(VI) from industrial wastewater was evaluated using a 2.0-m3 bioreactor. Liquid pineapple waste was used as a nutrient for the biofilm community formed inside the bioreactor. The use of rubber wood sawdust as packing material was able to immobilize more than 106 CFU mL−1 of Acinetobacter haemolyticus cells after 3 days of contact time. Complete reduction of 15–240 mg L−1 of Cr(VI) was achieved even after 3 months of bioreactor operation. Cr(VI) was not detected in the final effluent fraction indicating complete removal of Cr from solution from the flocculation/coagulation step and the unlikely re-oxidation of Cr(III) into Cr(VI). Impatiens balsamina L. and Gomphrena globosa L. showed better growth in the presence of soil–sludge mixture compared to Coleus scutellarioides (L.) Benth. Significant amounts of Cr accumulated at different sections of the plants indicate its potential application in Cr phytoremediation effort. The bacterial-based system was also determined not to be detrimental to human health based on the low levels of Cr detected in the hair and nail samples of the plant operators. Thus, it can be said that bacterial-based Cr(VI) treatment system is a feasible alternative to the conventional system especially for lower Cr(VI) concentrations, where sludge generated can be used as growth supplement for ornamental plant as well as not detrimental to the health of the workers.
Keywords: Bioremediation; Chromium; Bacteria; Pilot scale; Agriculture; Wastewater

The Cr6+-resistant plant growth-promoting bacteria was isolated from soil samples that were collected from an electroplating industry at Coimbatore, India, that had tolerated chromium concentrations up to 500 mg Cr6+/L in Luria-Bertani medium. Based on morphology, physiology, and biochemical characteristics, the strain was identified as Bacillus sp. following the Bergey's manual of determinative bacteriology. Evaluation of plant growth-promoting parameters has revealed the intrinsic ability of the strain for the production of indole-3-acetic acid (IAA), siderophore, and solubilization of insoluble phosphate. Bacillus sp. have utilized tryptophan as a precursor for their growth and produced IAA (122 μg/mL). Bacillus sp. also exhibited the production of siderophore that was tested qualitatively using Chrome Azurol S (CAS) assay solution and utilized the insoluble tricalcium phosphate as the sole source of phosphate exhibiting higher rate of phosphate solubilization after 72 h of incubation (1.45 μg/mL). Extent of Cr6+ uptake and accumulation of Cr6+ in the cell wall of Bacillus sp. was investigated using atomic absorption spectrophotometer and scanning electron microscope-energy dispersive spectroscopy, respectively. The congenital capability of this Cr6+-resistant plant growth-promoting Bacillus sp. could be employed as bacterial inoculum for the improvement of phytoremediation in heavy metal contaminated soils.
Keywords: PGPB; Chromium; IAA; Siderophore; Phosphate solubilization; SEM-EDAX

Multiplexed Detection of Waterborne Pathogens in Circular Microfluidics by Shailaja Agrawal; Amit Morarka; Dhananjay Bodas; K. M. Paknikar (1668-1677).
Microfluidic lab-on-a-chip presents an ideal solution for bacterial sensing and identification due to its advantages like large surface-to-volume ratio, requirement of low sample volume and multiplexing possibility. The present work deals with the development of an immunosensor chip using circular microchannels fabricated directly with microdimensional copper wire and permanent magnet for capture of Fe3O4 magnetic nanoparticle (MNP) conjugate. The MNP facilitate capture of the antigen in a confined space and hence, enhanced fluorescence signal for detection. The multiplexed microfluidic chip permits visual detection and quantification of waterborne pathogens viz. Escherichia coli and Salmonella typhimurium simultaneously. CdTe quantum dots (QDs) with different emission wavelengths were conjugated with anti-E. coli and anti-S. typhimurium antibodies for concurrent fluorescence detection. The present technique provides an inexpensive yet powerful tool to image and quantify pathogens at low numbers with passage of large sample volumes.
Keywords: 3D circular microchannels; E. coli ; S. typhimurium ; QDs

Effects of Sludge Concentrations and Different Sponge Configurations on the Performance of a Sponge-Submerged Membrane Bioreactor by Tien Thanh Nguyen; Huu Hao Ngo; Wenshan Guo; Jianxin Li; Andrzej Listowski (1678-1687).
The performance of a novel sponge-submerged membrane bioreactor (SSMBR) was evaluated to treat primary treated sewage effluent at three different activated sludge concentrations. Polyurethane sponge cubes with size of 1 × 1 × 1 cm were used as attached growth media in the bioreactor. The results indicated the successful removal of organic carbon and phosphorous with the efficiency higher than 98% at all conditions. Acclimatised sponge MBR showed about 5% better ammonia nitrogen removal at 5 and 10 g/L sludge concentration as compared to the new sponge system. The respiration test revealed that the specific oxygen uptake rate was around 1.0–3.5 mgO2/gVSS.h and likely more stable at 10 g/L sludge concentration. The sludge volume index of less than 100 mL/g during the operation indicated the good settling property of the sludge. The low mixed liquor suspended solid increase indicated that SSMBR could control the sludge production. This SSMBR was also successful in reducing membrane fouling with significant lower transmembrane pressure (e.g. only 0.5 kPa/day) compared to the conventional MBR system. Further study will be conducted to optimise other operating conditions.
Keywords: Sponge-submerged membrane bioreactor; Membrane fouling; Organic and nutrient removals; Activated sludge concentration

Assessment of Dentifrices Against Candida Biofilm by Nivedita Singh; Akhansha Nayyar; G. Bhattacharjee; A. K. Singh; Vikas Pruthi (1688-1698).
The invasion of opportunistic pleiomorphic Candida albicans into oral cavity environment leads to development and progression of its resistance to both naturally occurring antifungal peptides in human saliva as well as commercially available antifungal therapies. As a result of this, the usage and popularity of natural medicine and dentifrices had increased significantly in the last decade. In the present investigation, we have assessed the action of locally available dentifrices against C. albicans biofilm. Disk diffusion test showed maximum zone of inhibition (20 mm) by herbal dentifrice (D-5) as compared to other dentifrices when incubated at 37 °C and 48 h. Assessment of dentifrice D-5 for its effectiveness against C. albicans was further shown in MIC90 (3.12 mg mL−1) and SMIC90 (6.2 mg mL−1) values for planktonic and sessile cells (biofilm forming), respectively. Our data depicted 80% reduction in the cell surface hydrophobicity when 6.2 mg mL−1 of herbal dentifrice D-5 was used against 48-h grown Candida biofilm at 37 °C. Visualization of herbal dentifrice D-5-treated C. albicans biofilm under SEM revealed drastic reduction in the dense network of yeast, hyphae, and pseudohyphae enclosed in its ECM as compared to its control biofilm. The data were further supported by CLSM analysis which depicted C. albicans architecture disruption by herbal dentifrices. From the above data, it is inferred that these studies would provide researchers and medical practitioners with better insight into the antifungal effect of natural herbal dentifrices.
Keywords: Biofilm; Dentifrices; Candida albicans ; CLSM

Watermelon Rind: Agro-waste or Superior Biosorbent? by Cong Liu; Huu Hao Ngo; Wenshan Guo (1699-1715).
Biosorption of copper (Cu), zinc (Zn), and lead (Pb) on watermelon rind in a well-stirred batch system was investigated. pH showed significant influence on the biosorption process. Optimal pH for Cu, Zn, and Pb biosorption was found to be 5.0, 6.8 and 6.8, respectively. Watermelon rind was in favor of Pb and it could remove up to 99% Pb between pH ranges of 5 and 6.8 when Pb concentration is lower than 100 mg/L. The biosorptive capacity of watermelon on Cu, Zn, and Pb was 6.281, 6.845, and 98.063 mg/g, respectively. The equilibrium data fitted well to Langmuir adsorption isotherm while pseudo-second-order kinetic model exhibited more advantages for describing kinetic data than pseudo-first-order kinetic model. NaOH was found to be a suitable eluent. After desorption in NaOH solution, the resorption efficiency reached as high as 99% of these three metals either in a single-component or multi-component system. From the characterization study, ion exchange and micro-precipitation were estimated to be the main mechanisms. Due to its high metal uptake capacity, reusability, and metal recovery, watermelon rind can be considered as an eco-friendly and economic biosorbent for removing Pb from water and wastewater.
Keywords: Watermelon rind; Biosorption; Biosorbent; Heavy metal removal; Model; Desorption

Biofouling Potential Reductions Using a Membrane Hybrid System as a Pre-treatment to Seawater Reverse Osmosis by Sanghyun Jeong; Lan Hee Kim; Sung-Jo Kim; Tien Vinh Nguyen; Saravanamuthu Vigneswaran; In S. Kim (1716-1727).
Biofouling on reverse osmosis (RO) membranes is the most serious problem which affects desalination process efficiency and increases operation cost. The biofouling cannot be effectively removed by the conventional pre-treatment traditionally used in desalination plants. Hybrid membrane systems coupling the adsorption and/or coagulation with low-pressure membranes can be a sustainable pre-treatment in reducing membrane fouling and at the same time improving the feed water quality to the seawater reverse osmosis. The addition of powder activated carbon (PAC) of 1.5 g/L into submerged membrane system could help to remove significant amount of both hydrophobic compounds (81.4%) and hydrophilic compounds (73.3%). When this submerged membrane adsorption hybrid system (SMAHS) was combined with FeCl3 coagulation of 0.5 mg of Fe3+/L, dissolved organic carbon removal efficiency was excellent even with lower dose of PAC (0.5 g/L). Detailed microbial studies conducted with the SMAHS and the submerged membrane coagulation–adsorption hybrid system (SMCAHS) showed that these hybrid systems can significantly remove the total bacteria which contain also live cells. As a result, microbial adenosine triphosphate (ATP) as well as total ATP concentrations in treated seawater and foulants was considerably decreased. The bacteria number in feed water prior to RO reduced from 5.10E+06 cells/mL to 3.10E+03 cells/mL and 9.30E+03 cells/mL after SMAHS and SMCAHS were applied as pre-treatment, respectively. These led to a significant reduction of assimilable organic carbon (AOC) by 10.1 μg/L acetate-C when SMCAHS was used as a pre-treatment after 45-h RO operation. In this study, AOC method was modified to measure the growth of bacteria in seawater by using the Pseudomonas P.60 strain.
Keywords: Adenosine triphosphate (ATP); Assimilable organic carbon (AOC); Biofouling; Seawater reverse osmosis; Submerged membrane adsorption hybrid system; Submerged membrane coagulation–adsorption hybrid system

Fed-batch Anaerobic Valorization of Slaughterhouse By-products with Mesophilic Microbial Consortia Without Methane Production by J. Pessiot; R. Nouaille; M. Jobard; R. R. Singhania; A. Bournilhas; G. Christophe; P. Fontanille; P. Peyret; G. Fonty; C. Larroche (1728-1743).
This work aimed at setting up a fully instrumented, laboratory-scale bioreactor enabling anaerobic valorization of solid substrates through hydrogen and/or volatile fatty acid (VFA) production using mixed microbial populations (consortia). The substrate used was made of meat-based wastes, especially from slaughterhouses, which are becoming available in large amounts as a consequence of the growing constraints for waste disposal from meat industry. A reconstituted microbial mesophilic consortium without Archaebacteria (methanogens), named PBr, was cultivated in a 5-L anaerobic bioreactor on slaughterhouse wastes. The experiments were carried out with sequential fed-batch operations, including liquid medium removal from the bioreactor and addition of fresh substrate. VFAs and nitrogen were the main metabolites observed, while hydrogen accumulation was very low and no methane production was evidenced. After 1,300 h of culture, yields obtained for VFAs reached 0.38 g/g dry matter. Strain composition of the microbial consortium was also characterized using molecular tools (temporal temperature gradient gel electrophoresis and gene sequencing).
Keywords: Biohydrogen; Anaerobic mesophilic microorganisms; Slaughterhouse wastes; VFA; Nitrogen

Dimethyl sulfide (DMS) is one of the sulfurous pollutants present in the waste gas generated from the pulp and paper industry. DMS has environmental health implications; therefore, it is necessary to treat the waste gas containing DMS prior to discharge into the environment. A bench-scale biofilter was operated in the laboratory as well as in a pulp and paper industry for the treatment of DMS. Both the biofilters were packed with pre-sterilized wood chips and cow dung/compost of the same origin seeded with biomass developed from garden soil enriched with DMS. The biofilters were operated for the generation of process parameters, and the potential microorganisms isolated from both the biofilters have been purified and characterized for degradation of DMS. Further, these cultures were purified on a basal medium using DMS as a sole carbon source for the growth. Further, the purified cultures were characterized through standard fatty acid methyl esters (FAME)-gas chromatography method, and the isolates were found to be mesophilic, aerobic microbes. These microbes were identified as Bacillus sphaericus-GC subgroup F, Paenibacillus polymyxa, B. sphaericus-GC subgroup F, B. sphaericus-GC subgroup F, and Bacillus megaterium-GC subgroup A, respectively. The potential culture for degradation of DMS was identified as B. sphaericus by 16s rRNA molecular analysis.
Keywords: Biofilter; Wood chip; Cow dung; FAME profile; Viable count; 16s rRNA

Bioactives of Microbes Isolated from Western Ghat Belt of Kerala Show β-Lactamase Inhibition along with Wide Spectrum Antimicrobial Activity by Sowmya Pazhur Mohandas; Sita Ravikumar; Sumi J Menachery; Gayathri Suseelan; Sai Shyam Narayanan; Hemaraj Nandanwar; Kesavan Madhavan Nampoothiri (1753-1762).
The present study describes the exploitation of microbial biodiversity from Western Ghats of Kerala for screening of bioactives having β-lactamase inhibitory activities. A total of 700 pure cultures were isolated and were screened for antibacterial activity against a β-lactam resistant Bacillus cereus strain (PL 10) isolated from the same niche. Bioactive extracts made from 45 isolates showed inhibitory activities against PL 10, of which two strains showed inhibition of extended spectrum β-lactamase (ESBL) producing Klebsiella ESBL1101 and three strains inhibited methicillin-resistant Staphylococcus aureus (MRSA) strain MRSA831. All these five strains showed wide spectrum antimicrobial activity against various fungi and bacteria. These five cultures were identified by 16S rRNA sequencing and biochemical tests and the preliminary characterizations of their bioactive extracts were carried out. This study suggests the potential of bioactives from two inhibitor–producer strains, NII 167 and NII 1054, for being developed as inhibitors against wide spectrum β-lactam resistant strains.
Keywords: β-Lactam; β-Lactamase; Extended spectrum β-lactamases (ESBL); Bioactives; Biodiversity; Antimicrobial activity

Although it is known that Palk Bay sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their molecular diversity is still lacking. In the present study, bacterial diversity in Palk Bay sediments was characterized using the molecular method terminal-restriction fragment length polymorphisms (T-RFLP). The bacterial assemblages detected by T-RFLP analysis revealed that the nearshore sediment harbored high number of bacterial count, whereas the 2.5-m sediment harbored diverse and distinct bacterial composition with fine heterogeneity. The major bacterial groups detected in all the three sediment samples were Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria (including alpha (α), gamma (γ), delta (δ), and epsilon (ε)-Proteobacteria), and uncultured bacteria. This is the first study that reveals the presence of Bacteroidetes, delta (δ)- and epsilon (ε)-Proteobacteria, and uncultured bacteria in Palk Bay sediments. The hitherto unexplored wide microbial diversity of Palk Bay coastal area was unraveled in the current study through culture-independent approach. These data suggest that the continued use of cultivation-independent techniques will undoubtedly lead to the discovery of additional bacterial diversity and provide a direct means to learn more about the ecophysiology and biotechnological potential of Palk Bay coastal area.
Keywords: T-RFLP; Palk Bay; Marine sediments; 16S rRNA gene

Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens by Balu Jancy Kalpana; Subramonian Aarthy; Shunmugiah Karutha Pandian (1778-1794).
The extracellular α-amylase enzyme from Bacillus subtilis S8-18 of marine origin was proved as an antibiofilm agent against methicillin-resistant Staphylococcus aureus (MRSA), a clinical strain isolated from pharyngitis patient, Vibrio cholerae also a clinical isolate from cholera patient and Pseudomonas aeruginosa ATCC10145. The spectrophotometric and microscopic investigations revealed the potential of α-amylase to inhibit biofilm formation in these pathogens. At its BIC level, the crude enzyme caused 51.81–73.07% of biofilm inhibition. Beyond the inhibition, the enzyme was also effective in degradation of preformed mature biofilm by disrupting the exopolysaccharide (EPS), an essential component in biofilm architecture. Furthermore, the enzyme purified to its homogeneity by chromatographic techniques was also effective in biofilm inhibition (43.83–61.68%) as well as in degradation of EPS. A commercial α-amylase enzyme from B. subtilis was also used for comparative purpose. Besides, the effect of various enzymes and temperature on the antibiofilm activity of amylase enzymes was also investigated. This study, for the first time, proved that α-amylase enzyme alone can be used to inhibit/disrupt the biofilms of V. cholerae and MRSA strains and beholds much promise in clinical applications.
Keywords: α-Amylase; Bacillus subtilis S8-18; Biofilms; Confocal Laser Scanning Microscopy (CLSM); Exopolysaccharide (EPS)

Phytochemical Investigation of Bidens biternata (Lour.) Merr. and Sheriff.—A Nutrient-Rich Leafy Vegetable from Western Ghats of India by Pradeesh Sukumaran; Archana G. Nair; Devi M. Chinmayee; I. Mini; Swapna T. Sukumaran (1795-1801).
Bidens biternata, belonging to the family Asteraceae, is an erect annual herb, up to 1 cm in height, and a widespread weed of cultivated areas. This plant is common, particularly in the Western Ghats regions of Kerala state in India. It is used as a leafy vegetable by the Paniya and Kattunaayika tribes of Waynadu Districts in Kerala and also to cure hepatitis, cold, cough, dysentery, etc. The multiplication and utilization of this leafy vegetable will help to overcome the nutritional deficiency problem and also to maintain the biodiversity. For effective biochemical analysis, plant extract was taken using different solvents. Various phytochemicals like reducing sugar, glycosides, flavonoids, alkaloids, tannins, steroids, terpenoids, coumarins, saponins, anthraquinones, phlobatannins and iridoids were estimated. Different nutritional factors like total carbohydrates, total proteins, total reducing sugar, different amino acids, free fatty acids, crude fibre, lipids, total moisture content, vitamins, etc. were tested by standard estimation methods. Anti-nutritional factors like phytic acid, total phenol, tannic acid, etc., were also estimated. Micronutrients and different pigments were quantified. The present studies revealed that this wild leafy plant has numerous nutritional factors with a low level of anti-nutritional factors. Therefore, this nutritive herb with diverse health-promoting compounds can be effectively utilized to overcome the nutritional deficiency problem around the globe.
Keywords: Phytochemicals; Bidens biternata ; Nutritional factors; Anti-nutritional factors

The antioxidant properties of phenolic compounds from olive pulp (PCO) of chamlal variety and those of individual phenolic compounds were evaluated and compared with that of vitamin C (Vit C). The antioxidant activity was measured by the tests of iron reduction and scavenging hydrogen peroxide (H2O2). Results showed that all the substances tested exhibit a reducing power. The PCO present activities of iron reduction and H2O2 scavenging higher than those of Vit C. The protective effect of PCO against oxidation of lipids and proteins from erythrocyte membranes was studied. The measurement of malondialdehyde generated under oxidative stress conditions induced by hydroxyl radicals generating system revealed that PCO have the most significant protective activity against lipid peroxidation (IC50 = 49.27 ± 1.91 μg mL−1). Paradoxically, Vit C revealed a pro-oxidant effect. Proteins oxidation was evaluated using the H2O2/FeSO4 system and electrophoresis. In the presence of PCO at 1 mg mL−1, proteins of erythrocyte membranes were protected contrary to those treated with Vit C at the same concentration.
Keywords: Phenolic compounds; Olive; Antioxidant activities

In Vitro Propagation of Dioscorea alata var. purpurae by Heena J. Shah; S. S. Lele (1811-1817).
Dioscorea alata var. purpurae (Indian purple yam) is an important source of diosgenin, a triterpenoid that is used as a raw material in the synthesis of corticosteroid hormones. These drugs are used for the treatment of pharmacological conditions such as arthritis. This paper reports in vitro propagation of explants of various parts of Dioscorea—tuber, leaves, and nodes. Murashige and Skoog media supplemented with hormones and additives was used to get maximum callus initiation and shoot/root induction. All the cultures were maintained at 25 ± 2 °C under cool-white fluorescent tubes with 16-h photoperiod. Callus initiation was observed from 8th to 11th day of inoculation, and subsequent root/shoot was initiated in nodal callus after 21 days. Hormones such as kinetin, indole-3-acetic acid, indole-3-butyric acid, α-naphthalene acetic acid, and thiadizuron did not show significant enhancement. Also, there was no need for supplementing additives (silver nitrate, glutamine, l-asparagine monohydrate, polyethylene glycol). Combination of 6-benzylaminopurine (0.2 ppm) and 2,4-dichlorophenoxyacetic acid (2 ppm) hormones gave the best results, and all parts of the plants gave similar callus induction.
Keywords: Callus; Root and shoot; Dioscorea alata ; BAP; 2,4-D

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O2 in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O2 in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O2 in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).
Keywords: Hairy roots; Oxygen; Air-flow rate; Oxygen vectors; Azadirachtin; Growth; Productivity

Development of a Mathematical Model for Growth and Oxygen Transfer in In Vitro Plant Hairy Root Cultivations by Rajashekar Reddy Palavalli; Smita Srivastava; Ashok Kumar Srivastava (1831-1844).
Genetically transformed, “Hairy roots” once developed can serve as a stable parent culture for in vitro production of plant secondary metabolites. However, the major bottleneck in the commercial exploitation of hairy roots remains its successful scale-up due to oxygen transfer limitation in three-dimensionally growing hairy root mass. Mass transfer resistances near the gas–liquid and liquid–solid boundary layer affect the oxygen delivery to the growing hairy roots. In addition, the diffusional mass transfer limitation due to increasing size of the root ball (matrix) with growth also plays a limiting role in the oxygen transfer rate. In the present study, a mathematical model is developed which describes the oxygen transfer kinetics in the growing Azadirachta indica hairy root matrix as a case study for offline simulation of process control strategies ensuring non-limiting concentrations of oxygen in the medium throughout the hairy root cultivation period. The unstructured model simulates the effect of oxygen transfer limitation in terms of efficiency factor (η) on specific growth rate (μ) of the hairy root biomass. The model is able to predict effectively the onset of oxygen transfer limitation in the inner core of the growing hairy root matrix such that the bulk oxygen concentration can be increased so as to prevent the subsequent inhibition in growth of the hairy root biomass due to oxygen transfer (diffusional) limitation.
Keywords: Hairy roots; Oxygen transfer rate; Specific growth rate; Efficiency factor; Mathematical model; Azadirachta indica