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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #4)

Kinetic Analysis and Modeling of Daptomycin Batch Fermentation by Streptomyces roseosporus by Wenyu Lu; Jinghua Fan; Jianping Wen; Zhendong Xia; Qinggele Caiyin (pp. 453-462).

In this study, Streptomyces roseosporus was subjected to helium–neon (He–Ne) laser (632.8 nm) irradiation to improve the production ability of extracellular antibiotic daptomycin. Under the optimum irradiation dosage of 18 mW for 22 min, a stable positive mutant strain S. roseosporus LC-54 was obtained. The maximum A21978C (daptomycin is a semisynthetic antimicrobial substance derived from the A21978C complex) yield of this mutant strain was 296 mg/l, which was 146% higher than that of the wild strain. The mutant strain grew more quickly and utilized carbohydrate sources more efficiently than the wild strain. The batch culture kinetics was investigated in a 7 l bioreactor. The logistic equation for growth, the Luedeking–Piret equation for daptomycin production, and Luedeking–Piret-like equations for carbon substrate consumption were established. This model appeared to provide a reasonable description for each parameter during the growth phase and fitted fairly well with the experiment data.

Keywords: Daptomycin; Streptomyces roseosporus ; Batch culture; Laser irradiation; Kinetic model

Studies on Characterization of Bioflocculant Exopolysaccharide of Azotobacter indicus and Its Potential for Wastewater Treatment by Satish Vitthalrao Patil; Chandrashekhar D. Patil; Bipinchandra K. Salunke; Rahul B. Salunkhe; G. A. Bathe; Deepak M. Patil (pp. 463-472).

Partially characterized bioflocculant exopolysaccharide (EPS) produced from an Azotobacter indicus ATCC 9540 strain reported in our previous study was further characterized, and its flocculant potential was investigated at different pH, temperature, and cations concentrations. Flocculant activity at different concentrations of EPS in the absence of cations was reanalyzed by slight modified flocculant assay. It revealed that flocculant activity increased in a concentration-dependent manner up to a certain limit, with the maximum flocculation of 72% at 500 mgL⁻¹ EPS concentration, even in the absence of cations. At the concentration of 10 mgL⁻¹, CaCl₂ showed more significant activity (92%) than AlCl₃ and MnSO₄. Differential scanning calorimetry study and flocculant assay revealed high temperature stability of EPS up to 97 °C. Molecular weight of the EPS determined by size exclusion chromatography was found to be approximately 2 × 10⁶ kDa. Investigation on flocculation efficacy of the characterized EPS for wastewater treatment of dairy, woolen, starch, and sugar industry suggested it to be effective and stable at wide pH range of 5–10. Wastewater treatment with biopolymer at 500 mgL⁻¹ showed reduction in biochemical oxygen demand (38–80%), chemical oxygen demand (37–79%), and suspended solids (41–68%). This study suggests that Azotobacter polymer has high potential in wastewater treatment as bioflocculant and can be used as a potential alternative to chemical flocculants.

Keywords: Bioflocculant; Waste treatment; DSC; Suspended solids; Exopolysaccharide

Poly-β-hydroxybutyrate and Exopolysaccharide Biosynthesis by Bacterial Isolates from Pigeonpea [Cajanus cajan (L.) Millsp] Root Nodules by Paulo Ivan Fernandes Júnior; Paulo Jansen de Oliveira; Norma Gouvêa Rumjanek; Gustavo Ribeiro Xavier (pp. 473-484).

The bacterial strains that are able to produce biopolymers that are applied in industrial sectors present a source of renewable resources. Some microorganisms are already applied at several industrial sectors, but the prospecting of new microbes must bring microorganisms that are feasible to produce interesting biopolymers more efficiently and in cheaper conditions. Among the biopolymers applied industrially, polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) stand out because of its applications, mainly in biodegradable plastic production and in food industry, respectively. In this context, the capacity of bacteria isolated from pigeonpea root nodules to produce EPS and PHB was evaluated, as well as the cultural characterization of these isolates. Among the 38 isolates evaluated, the majority presented fast growth and ability to acidify the culture media. Regarding the biopolymer production, five isolates produced more than 10 mg PHB per liter of culture medium. Six EPS producing bacteria achieved more than 200 mg EPS per liter of culture medium. Evaluating different carbon sources, the PHB productivity of the isolate 24.6b reached 69% of cell dry weight when cultured with starch as sole carbon source, and the isolate 8.1c synthesized 53% PHB in dry cell biomass and more than 1.3 g L⁻¹ of EPS when grown using xylose as sole carbon source.

Keywords: Biopolymers; Rhizobia; UV-HPLC; Technological innovation; Inoculant technology; Strain selection

Adaptive Evolution of Escherichia coli Inactivated in the Phosphotransferase System Operon Improves Co-utilization of Xylose and Glucose Under Anaerobic Conditions by Victor Emmanuel Balderas-Hernández; Verónica Hernández-Montalvo; Francisco Bolívar; Guillermo Gosset; Alfredo Martínez (pp. 485-496).

Modification of the phosphoenolpyruvate/sugar phosphotransferase system (PTS) has shown improvement in sugar coassimilation in Escherichia coli production strains. However, in preliminary experiments under anaerobic conditions, E. coli strains with an inactive PTS and carrying pLOI1594, which encodes pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, were unable to grow. These PTS⁻ strains were previously evolved under aerobic conditions to grow rapidly in glucose (PTS^- Glucose⁺ phenotype). Thus, in this work, applying a continuous culture strategy under anaerobic conditions, we generate a new set of evolved PTS⁻ Glucose⁺ mutants, VH30N1 to VH30N6. Contrary to aerobically evolved mutants, strains VH30N2 and VH30N4 carrying pLOI1594 grew in anaerobiosis; also, their growth capacity was restored in a 100%, showing specific growth rates (μ ~ 0.12 h⁻¹) similar to the PTS⁺ parental strain (μ = 0.11 h⁻¹). In cultures of VH30N2/pLOI1594 and VH30N4/pLOI1594 using a glucose–xylose mixture, xylose was totally consumed and consumption of sugars occurred in a simultaneous manner indicating that catabolic repression is alleviated in these strains. Also, the efficient sugar coassimilation by the evolved strains caused an increment in the ethanol yields.

Keywords: Anaerobiosis; Evolved strains; PTS; Sugar co-utilization; Xylose

Biodegradation of Chlorinated and Non-chlorinated VOCs from Pharmaceutical Industries by P. Balasubramanian; Ligy Philip; S. Murty Bhallamudi (pp. 497-518).

Biodegradation studies were conducted for major organic solvents such as methanol, ethanol, isopropanol, acetone, acetonitrile, toluene, chloroform, and carbon tetrachloride commonly used in pharmaceutical industries. Various microbial isolates were enriched and screened for their biodegradation potential. An aerobic mixed culture that had been previously enriched for biodegradation of mixed pesticides was found to be the most effective. All the organic solvents except chloroform and carbon tetrachloride were consumed as primary substrates by this mixed culture. Biodegradation rates of methanol, ethanol, isopropanol, acetone, acetonitrile, and toluene were measured individually in batch systems. Haldane model was found to best fit the kinetics of biodegradation. Biokinetic parameters estimated from single-substrate experiments were utilized to simulate the kinetics of biodegradation of mixture of substrates. Among the various models available for simulating the kinetics of biodegradation of multi-substrate systems, competitive inhibition model performed the best. Performance of the models was evaluated statistically using the dimensionless modified coefficient of efficiency (E). This model was used for simulating the kinetics of biodegradation in binary, ternary, and quaternary substrate systems. This study also reports batch experiments on co-metabolic biodegradation of chloroform, with acetone and toluene as primary substrates. The Haldane model, modified for inhibition due to chloroform, could satisfactorily predict the biodegradation of primary substrate, chloroform, and the microbial growth.

Keywords: Biodegradation; Substrate inhibition kinetics; Organic solvents; Volatile organic compounds; Pharmaceutical industry

Endpoint Fragmentation Index: A Method for Monitoring the Evolution of Microbial Degradation of Polysaccharide Feedstocks by Terrence R. Green; Radu Popa (pp. 519-527).

We describe a simple method for tracking the course of microbial degradation of polysaccharide-rich feedstocks. The method involves determining total polysaccharides present in the feedstock, measured in glucose equivalents, relative to the fractional component of polysaccharides exhibiting 2,3-dinitrosalycylic acid aldehyde activity. The ratio of total polysaccharide to aldehyde activity, defined as the end-point fragmentation (EPF) index, is then calculated and tracked as it shifts as microbial degradation of polysaccharide-rich feedstock progresses. While degradation occurs, the EPF index falls. It bottoms out at an asymptotic limit marking the point in time where further degradation of the polysaccharide-rich feedstock has ceased. The EPF index can be used to follow the progressive breakdown of composting polysaccharide-rich waste. It may also have applicability as a means of tracking the turnover of polysaccharides in other complex environments including soil, sediments, wetlands, and peat bogs.

Keywords: Cellulose; Polysaccharides; EPF index; Compost

A Specific, Robust, and Automated Method for Routine At-Line Monitoring of the Concentration of Cellulases in Genetically Modified Sugarcane Plants by Ruchi Gupta; Sara J. Baldock; Peter R. Fielden; Bruce D. Grieve (pp. 528-539).

Bagasse is one of the waste crop materials highlighted as commercially viable for cellulosic bio-ethanol production via enzymatic conversion to release fermentable sugars. Genetically modified sugarcane expressing cellobiohydrolases (CBH), endoglucanase (EG), and β-glucosidases (BG) provide a more cost-effective route to cellulose breakdown compared to culturing these enzymes in microbial tanks. Hence, process monitoring of the concentration profile of these key cellulases in incoming batches of sugarcane is required for fiscal measures and bio-ethanol process control. The existing methods due to their non-specificity, requirement of trained analysts, low sample throughput, and low amenability to automation are unsuitable for this purpose. Therefore, this paper explores a membrane-based sample preparation method coupled to capillary zone electrophoresis (CZE) to quantify these enzymes. The maximum enzyme extraction efficiency was obtained by using a polyethersulfone membrane with molecular cut-off of 10 kDa. The use of 15 mM, pH 7.75, phosphate buffer resulted in CZE separation and quantification of CBH, EG, and BG within 10 min. Migration time reproducibility was between 0.56% and 0.7% and hence, suitable for use with automatic peak detection software. Therefore, the developed CZE method is suitable for at-line analysis of BG, CBH, and EG in every batch of harvested sugarcane.

Keywords: Bio-fuel; Sugarcane; Enzyme expression; Process monitoring; Capillary zone electrophoresis

Replacing Synthetic with Microbial Surfactants as Collectors in the Treatment of Aqueous Effluent Produced by Acid Mine Drainage, Using the Dissolved Air Flotation Technique by Carlyle T. B. Menezes; Erilson C. Barros; Raquel D. Rufino; Juliana M. Luna; Leonie A. Sarubbo (pp. 540-546).

Dissolved air flotation (DAF) is a well-established separation process employing micro bubbles as a carrier phase. The application of this technique in the treatment of acid mine drainage, using three yeast biosurfactants as alternative collectors, is hereby analyzed. Batch studies were carried out in a 50-cm high acrylic column with an external diameter of 2.5 cm. High percentages (above 94%) of heavy metals Fe(III) and Mn(II) were removed by the biosurfactants isolated from Candida lipolytica and Candida sphaerica and the values were found to be similar to those obtained with the use of the synthetic sodium oleate surfactant. The DAF operation with both surfactant and biosurfactants, achieved acceptable turbidity values, in accordance with Brazilian standard limits. The best ones were obtained by the biosurfactant from C. lipolytica, which reached 4.8 NTU. The results obtained with a laboratory synthetic effluent were also satisfactory. The biosurfactants removed almost the same percentages of iron, while the removal percentages of manganese were slightly higher compared with those obtained in the acid mine drainage effluent. They showed that the use of low-cost biosurfactants as collectors in the DAF process is a promising technology for the mining industries.

Keywords: Biosurfactants; Flotation; Effluent treatment; Heavy metals; Candida ; Low-cost substrate

Dilute Acid Pretreatment of Black Spruce Using Continuous Steam Explosion System by Haixia Fang; James Deng; Tony Zhang (pp. 547-557).

The pretreatment of lignocellulosic materials prior to the enzymatic hydrolysis is essential to the sugar yield and bioethanol production. Dilute acid hydrolysis of black spruce softwood chip was performed in a continuous high temperature reactor followed with steam explosion and mechanical refining. The acid-soaked wood chips were pretreated under different feeding rates (60 and 92 kg/h), cooking screw rotation speeds (7.2 and 14.4 rpm), and steam pressures (12 and 15 bar). The enzymatic hydrolysis was carried out on the acid-insoluble fraction of pretreated material. At lower feeding rate, the pretreatment at low steam pressure and short retention time favored the recovery of hemicellulose. The pretreatment at high steam pressure and longer retention time recovered less hemicellulose but improved the enzymatic accessibility. As a result, the overall sugar yields became similar no matter what levels of the retention time or steam pressure. Comparing with lower feeding rate, higher feeding rate resulted in consistently higher glucose yield in both liquid fraction after pretreatment and that released after enzymatic hydrolysis.

Keywords: Dilute acid pretreatment; Enzymatic hydrolysis; Softwood; Continuous steam explosion; Refining

Plasma-Assisted Pretreatment of Wheat Straw by Nadja Schultz-Jensen; Frank Leipold; Henrik Bindslev; Anne Belinda Thomsen (pp. 558-572).

O₃ generated in a plasma at atmospheric pressure and room temperature, fed with dried air (or oxygen-enriched dried air), has been used for the degradation of lignin in wheat straw to optimize the enzymatic hydrolysis and to get more fermentable sugars. A fixed bed reactor was used combined with a CO₂ detector and an online technique for O₃ measurement in the fed and exhaust gas allowing continuous measurement of the consumption of O₃. This rendered it possible for us to determine the progress of the pretreatment in real time (online analysis). The process time can be adjusted to produce wheat straw with desired lignin content because of the online analysis. The O₃ consumption of wheat straw and its polymeric components, i.e., cellulose, hemicellulose, and lignin, as well as a mixture of these, dry as well as with 50% water, were studied. Furthermore, the process parameters dry matter content and milled particle size (the extent to which the wheat straw was milled) were investigated and optimized. The developed methodology offered the advantage of a simple and relatively fast (0.5–2 h) pretreatment allowing a dry matter concentration of 45–60%. FTIR measurements did not suggest any structural effects on cellulose and hemicellulose by the O₃ treatment. The cost and the energy consumption for lignin degradation of 100 g of wheat straw were calculated.

Keywords: Pretreatment method; Wheat straw; Lignin; Plasma; Discharge; Biofuel; Sustainable energy; Ozonisation

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #4)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #4)