Proton transport inside the biofilm limits electrical current generation by anode-respiring bacteria

Anode-respiring bacteria (ARB) in a biofilm anode carry out an oxidation half-reaction of organic matter, producing an electrical current from renewable biomass, including wastes. At the same time, ARB produce protons, usually one proton for every electron. Our study shows how current density generated by an acclimated ARB biofilm was limited by proton transport out of the biofilm. We determined that, at high current densities, protons were mainly transported out of the biofilm by protonating the conjugate base of the buffer system; the maximum current generation was directly related to the transport of the buffer, mainly by diffusion, into and out of the biofilm. With non-limiting acetate concentrations, the current density increased with higher buffer concentrations, going from 2.21 +/- 0.02 A m(-2) with 12.5-mM phosphate buffer medium to 9.3 +/- 0.4 A m(-2) using a 100-mM phosphate buffer at a constant anode potential of E(anode) = -0.35 V versus Ag/AgCl. Increasing the concentration of sodium chloride in the medium (0-100 mM) increased current density by only 15%, indicating that ion migration was not as important as diffusion of phosphate inside the biofilm. The current density also varied strongly with medium pH as a result of the buffer speciation: The current density was 10.0 +/- 0.8 A m(-2) at pH 8, and the pH giving one-half the maximum rate was 6.5. A j-V curve analysis using 100 mM phosphate buffer showed a maximum current density of 11.5 +/- 0.9 A m(-2) and half-saturation potential of -0.414 V versus Ag/AgCl, a value that deviated only slightly from the standard acetate potential, resulting in small anode-potential losses. We discuss the implications of the proton-transport limitation in the field of microbial fuel cells and microbial electrolytic cells.     

油脂酵母发酵木糖生产微生物油脂

目的用斯达油脂酵母(Lipomyces starkeyi)作为发酵菌株,以纯木糖溶液为油脂发酵原料,对L.starkeyi利用木糖积累油脂进行系统研究。方法 L.starkeyi于斜面培养基中活化后,接种于YPD液体培养基,于30℃、200 r/min摇床培养。在摇瓶中培养一段时间后,测定发酵液细胞浓度,离心发酵液收集细胞。将离心后得到的菌体加入木糖溶液重悬,并转接于含50 mL木糖溶液的250 mL摇瓶中进行发酵生产。结果相比一阶段法,两阶段发酵方法可以在更短的时间内达到较高的油脂含量,油脂含量能够达到细胞自身干重的60%以上。实验发现高菌龄酵母产油速度更快;并且初始木糖浓度高达120 g/L时,酵母细胞仍然能够高效合成油脂。结论 L.starkeyi能够有效利用木糖进行发酵产生油脂,是以木质纤维素为原料生产微生物油脂的优良菌种。     

Enhancement of coulombic efficiency and salt tolerance in microbial fuel cells by graphite/alginate granules immobilization of Shewanella oneidensis MR-1

Coulombic efficiency and stability of electricity output are crucial for practical applications of microbial fuel cells (MFCs). In this study, a cell immobilization method for electrogenic microorganism in MFCs using graphite/alginate granules is developed. The MFC with immobilized cell granules delivered a much more stable electricity output than that with suspension cells, and resulted in a ～0.8 to 1.7 times improvement on coulombic efficiency compared to the suspension mode. Impressively, with the conductive graphite/alginate/cells granules, the internal resistance of the MFC decreased dramatically. Moreover, the cell immobilized MFC showed a much higher tolerance to the shock of high salt concentration than the MFC with suspension cells. The results substantiated that immobilization of electrogenic microorganism for MFCs could be achieved by the method developed here, and it is promising for practical application in energy harvesting from wastewater by MFCs.     

Electricity production from xylose in fed-batch and continuous-flow microbial fuel cells

A medium-scale (0.77 l) air-cathode, brush-anode microbial fuel cell (MFC) operated in fed-batch mode using xylose (20 mM) generated a maximum power density of 13 +/- 1 W/m(3) (673 +/- 43 mW/m(2)). Xylose was rapidly removed (83.5%) within 8 h of a 60-h cycle, with 42.1% of electrons in intermediates (8.5 +/- 0.2 mM acetate, 5.9 +/- 0.01 mM ethanol, 4.3 +/- 0.1 mM formate, and 1.3 +/- 0.03 mM propionate), 9.1% captured as electricity, 16.1% in the remaining xylose, and 32.7% lost to cell storage, biomass, and other processes. The final Coulombic efficiency was 50%. At a higher initial xylose concentration (54 mM), xylose was again rapidly removed (86.9% within 24 h of a 116-h cycle), intermediates increased in concentration (18.4 +/- 0.4 mM acetate, 7.8 +/- 0.4 mM ethanol and 2.1 +/- 0.2 mM propionate), but power was lower (5.2 +/- 0.4 W/m(3)). Power was increased by operating the reactor in continuous flow mode at a hydraulic retention time of 20 h (20 +/- 1 W/m(3)), with 66 +/- 1% chemical oxygen demand removal. These results demonstrate that electricity generation is sustained over a cycle primarily by stored substrate and intermediates formed by fermentation and that the intermediates produced vary with xylose loading.     

Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

枯草芽孢杆菌微生态制剂发酵研究进展

微生态制剂是饲用抗生素的绿色有效替代品。枯草芽孢杆菌在逆境中可形成抗逆性强的芽孢,在生产和应用过程中保持高活性,是一种高效的微生态制剂菌种。提高枯草芽孢杆菌活菌数及芽孢率是保证微生态制剂产品质量的关键。本文综述了枯草芽孢杆菌芽孢形成的分子生物学机制及影响芽孢形成的重要因素,进一步比较枯草芽孢杆菌微生态制剂不同发酵方式的特点,重点阐述了提高枯草芽孢杆菌有效生物量的工艺优化,最后介绍了枯草芽孢杆菌微生态制剂的应用,并对将来研究思路进行了讨论。     

Effect of organic loading and retention time on dairy manure fermentation

The investigations presented and discussed herein establish an enhanced understanding on volatile fatty acid (VFA) production as a function of dairy manure fermenter organic loading (OL) and retention time (RT), first through a factorial of 64 fermentation potential (FP) batch tests, followed by analysis of a continuously operated pilot-scale fermenter. The maximum observed net FP - 0.103 mg VFA produced (as COD) (mg VS applied)⁻¹ - occurred at an OL of 40.7 g VS L⁻¹ and at a RT of 6 days. The pilot-scale fermenter exhibited an average yield of 0.09 mg VFA (as COD) synthesized (mg VS applied)⁻¹, with average effluent total VFA concentrations of 6398 mg VFA (as COD) L⁻¹. The research demonstrates that FP tests are an effective method to optimize continuously operated dairy manure fermenters, and that dairy manure fermentation can yield large quantities of organic acids at short RTs and high OL rates.     

Operation of a bioelectrochemical system as a polishing stage for the effluent from a two-stage biohydrogen and biomethane production process

Anaerobic bioenergy production processes including fermentative biohydrogen (BioH₂), anaerobic digestion (AD) and bioelectrochemical system have been investigated for converting municipal waste or various biomass feedstock to useful energy carriers. However, the performance of a microbial fuel cell (MFC) fed on the effluent from a two-stage biogas production process has not yet been investigated extensively in continuous reactor operation on complex substrates. In this study we have investigated the extent to which a microbial fuel cell (MFC) can reduce COD and recover further energy from the effluent of a two-stage biohydrogen and biomethane system. The performance of a four-module tubular MFC was determined at six different organic loadings (0.036–6.149 g sCOD L⁻¹ d⁻¹) in terms of power generation, COD removal efficiency, coulombic efficiency (CE) and energy conversion efficiency (ECE). A power density of 3.1 W m⁻³ was observed at the OLR = 0.572 g sCOD L⁻¹ d⁻¹, which resulted in the highest CE (60%) and ECE (0.8%), but the COD removal efficiency decreased at higher organic loading rates (35.1–4.4%). The energy recovery was 92.95 J L⁻¹ and the energy conversion efficiency, based on total influent COD was found to be 0.48–0.81% at 0.572 g sCOD L⁻¹ d⁻¹. However, the energy recovery by the MFC is only reported for a four-module reactor and improved performance can be expected with an extended module count, as chemical energy remained available for further electrogenesis.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

微生物发酵产木聚糖酶研究进展

木聚糖是植物半纤维素的主要成分,是自然界中仅次于纤维素的可再生资源。木聚糖酶是一类重要的木糖苷键水解酶酶系,可将木聚糖逐次降解为低聚木糖及木糖,在饲料、造纸、食品和生物转化等行业应用广泛。目前利用微生物发酵生产木聚糖酶的研究很多,菌种涉及到细菌、真菌等,其发酵生产木聚糖酶的工艺、产量及特性也各有不同,对此进行了综述,并展望了木聚糖酶发酵生产的研究方向。     

Comparative studies on fermentation products of cocoa beans

The maximum amounts of acetic acid produced by ripe and unripe cocoa beans were 157 mg and 110 mg/10 g wet wt of cotyledon, respectively. The unripe beans had a lower pH than the ripe beans after 6 days' fermentation. About 40% of ripe beans achieved a chocolate colour compared with 27% of unripe beans.O. Abdul Samah, N. Ibrahim and H. Alimon are with the Department of Biochemistry and Microbiology and M.I. Abdul Karim is with the Department of Food Science, both of the University of Agriculture, Malaysia, 43400 Serdang, Malaysia     

Effects of biofouling on ion transport through cation exchange membranes and microbial fuel cell performance

This study examines the effects of biofouling on the electrochemical properties of cation exchange membranes (CEMs), such as membrane electrical resistance (MER), specific proton conductivity (SC), and ion transport number (t₊), in addition to on microbial fuel cell (MFC) performance. CEM biofouling using a 15.5 ± 4.6 μm biofilm was found to slightly increase the MER from 15.65 Ω cm² (fresh Nafion) to 19.1 Ω cm², whereas an increase of almost two times was achieved when the electrolyte was changed from deionized water to an anolyte containing a high cation concentration supporting bacterial growth. The simple physical cleaning of CEMs had little effect on the Coulombic efficiency (CE), whereas replacing a biofouled CEM with new one resulted in considerable increase of up to 59.3%, compared to 45.1% for a biofouled membrane. These results clearly suggest the internal resistance increase of MFC was mainly caused by the sulfonate functional groups of CEM being occupied with cations contained in the anolyte, rather than biofouling itself.     

Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation

The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A–E when grown on agar bearing moist polyester–cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A–E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester–cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery.     

Effect of the proportions of neutral detergent fibre and starch, and their degradation rates, on in vitro ruminal fermentation

Effects of proportions of neutral detergent fibre (aNDFom) and starch, as well as their degradation rates, on rumen fermentation were tested using an in vitro rumen simulation system (SIMCO). The in vitro system was designed to simulate selective particle retention and had an average fluid volume of 1150 ml with a liquid dilution rate of approximately 0.07 h⁻¹. Two types of hay (aNDFom sources) and two types of starch were each included at two different levels in the diet and were examined in an experiment following a 2×2×2 factorial arrangement of treatments (eight diet combinations). The hay was either late-cut timothy (Phleum pretense L.) or early cut meadow grass (Poa pratensis L.), with ruminal in situ aNDFom digestion rates of 0.03–0.04 and 0.07–0.08 h⁻¹, respectively. The two starch types were raw (R) and cooked (C) potato starch with previously determined in vitro ruminal digestion rates of 0.04 and 0.20 h⁻¹, respectively. The starch levels were 300 and 600 g/kg diet dry matter (DM) with the remaining being hay (282–682 g/kg DM) and peptone (14–111 g/kg DM). The aNDFom level varied among the diets with different starch levels and hay types. The peptone acted as a source of peptides and, together with ammonia salts from buffer, was used to balance the N contents of the diets. The feeding level for each of the eight vessels was 28 g DM/d. Two 10-day simulations were made with the system. The average pH was higher (P<0.05) for all treatments with raw potato starch (6.19) versus cooked starch (6.07). Protozoa scores, on a qualitative scale, declined faster at the higher starch level. The aNDFom digestibility was, as expected, higher (P<0.001) for meadow hay (0.57) than timothy (0.32), and was also higher (P<0.001) at the lower starch level (0.54) versus the higher (0.35). Microbial protein production efficiency (mg microbial N/g organic matter truly digested) was higher for the faster degrading aNDFom (P<0.01) and starch (P<0.05) sources, but was not affected by starch level. Cooked starch resulted in a lower acetate proportion (449 mmol/mol versus 591 mmol/mol VFA; P<0.001) but higher proportions of propionate (297 mmol/mol versus 236 mmol/mol VFA; P<0.001), and butyrate (169 mmol/mol versus 127 mmol/mol VFA; P<0.01). Butyrate increased with starch level (127 mmol/mol versus 169 mmol/mol VFA; P<0.01), and was also higher for meadow hay versus timothy (168 mmol/mol versus 128 mmol/mol VFA; P<0.01). Interactions between the treatments demonstrate that the response in VFA pattern to starch level is dependent on starch and aNDFom sources. Substrates such as starch and aNDFom are fermented differently depending on their rates of ruminal degradation.     

Excretion of fermentation products in dark and anaerobically incubated cyanobacteria

An arbitrarily chosen selection of 37 cyanobacterial strains of the Oldenburg culture collection were tested for their ability of fermentation and secretion of fermentation products. In all examined strains at least one fermentation product could be detected. For the most part fermentation products were only shed in traces. Thus, for a large part of the investigated strains fermentation does not seem to be a sufficient metabolism to survive dark and anaerobic periods. Only five strains secreted remarkable amounts of products. Glycollate was mostly found in combination with formate and/or traces of oxalate. Lactate, ethanol and acetate were found in combination or single. Most of those strains sheding high amounts of glycollate and formate, did not show a remarkable lactate, ethanol or acetate excretion; those excreting high amounts of lactate, ethanol or acetate produced only minor volumes of glycollate and formate. It was not possible to find similar fermentation patterns by comparing fermentation of species belonging to the same family. Organisms fermenting or not fermenting could be found among marine, brackish and freshwater cyanobacteria. Fermentation, therefore seems to be a unique, and likely old capability among cyanobacteria, which was partly lost during evolution.     

Electricity generation by anaerobic bacteria and anoxic sediments from hypersaline soda lakes

Anaerobic bacteria and anoxic sediments from soda lakes produced electricity in microbial fuel cells (MFCs). No electricity was generated in the absence of bacterial metabolism. Arsenate respiring bacteria isolated from moderately hypersaline Mono Lake (Bacillus selenitireducens), and salt-saturated Searles Lake, CA (strain SLAS-1) oxidized lactate using arsenate as the electron acceptor. However, these cultures grew equally well without added arsenate using the MFC anode as their electron acceptor, and in the process oxidized lactate more efficiently. The decrease in electricity generation by consumption of added alternative electron acceptors (i.e. arsenate) which competed with the anode for available electrons proved to be a useful indicator of microbial activity and hence life in the fuel cells. Shaken sediment slurries from these two lakes also generated electricity, with or without added lactate. Hydrogen added to sediment slurries was consumed but did not stimulate electricity production. Finally, electricity was generated in statically incubated “intact” sediment cores from these lakes. More power was produced in sediment from Mono Lake than from Searles Lake, however microbial fuel cells could detect low levels of metabolism operating under moderate and extreme conditions of salt stress.     

The decomposition of tylosin fermentation waste in soil at four temperatures

Summary Aerobic decomposition of tylosin fermentation waste was studied by O₂ uptake and CO₂, NH₄ ⁺ and NO₃ ^– release over 10 weeks in a light compost-soil at 3 concentrations and 4 temperatures. Comparisons of O₂ uptake and CO₂ release at each temperature showed that aerobic conditions were maintained in the system. Maximal rates of respiration (C mineralization) increased with temperature. At 23°C 50% of the substrate C had been mineralized in 10 weeks. At 10–15°C and at 4°C C mineralization was approximately 38% and 22% respectively. Except at 4°C mineralization had almost ceased within 10 weeks. There was evidence of a permanent inhibition of C mineralization at 10–15°C compared with 23°C, and a temporary inhibition at 10°C compared with 15°C.At 10 weeks 25% of the N had been mineralized at 23, 15 and 10°C, while 14% had been mineralized at 4°C. The time taken to reach maximum N mineralization was reduced by increase in temperature and by 10 weeks mineralization had almost ceased at 15 and 23°C. In terms of the fertilizing effect of tylosin fermentation, 25% of the total N was available within 10 weeks at 10–23°C. Nitrification was strongly inhibited at 4 and 10°C. Both C and N mineralization were in direct proportion to the concentration of tylosin fermentation waste added to the soil.     

试论肠菌与中草药联合和发酵防治疾病

【摘 要】 本文阐述了肠道原始固有菌群（肠菌）与天然药用植物（中草药）联合应用和用肠菌发酵中草药的可行性及应用前景。     

非常规酵母天然产物合成

以解脂耶氏酵母(Yarrowia lipolytica)、巴斯德毕赤酵母(Pichia pastoris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、圆红冬孢酵母(Rhodosporidium toruloides)、多形汉逊酵母(Hansenula polymorpha)为代表的非常规酵母凭借较广的底物利用谱、较强的环境耐受性等优势,已成功实现多种天然产物的高效生产。随着合成生物学及基因编辑技术的发展,针对非常规酵母代谢工程改造的工具和策略也逐渐丰富。本文介绍了几类常见的非常规酵母的生理特性、工具开发及应用现状,并总结归纳了天然产物合成优化中常用的代谢工程策略;最后讨论了现阶段非常规酵母作为天然产物合成细胞工厂的优势和不足,并对后续研究和发展趋势进行了展望。     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.