Penicillium strains as dominant degraders in soil for coffee residue, a biological waste unsuitable for fertilization

AIMS: Coffee residue is an agricultural waste which inhibits the growth of several crops. Therefore coffee residue-degrading microbes in soil were screened, isolated and characterized. METHODS AND RESULTS: Forty isolates were obtained after enrichment culture of soil samples. Seven strains (fast degraders) showed strong degrading activity, while 18 strains (slow degraders) showed weak degrading activity. DNA analysis suggested that the fast degraders are Penicillium, and the slow degraders are Penicillium, Trichoderma/Hypocrea, Fusarium/Gibberella, Phaeoacremonium/Togninia or Acidocella. The all fast degraders are cellulolytic, mannolytic and pectinolytic. CONCLUSIONS: Although it is generally thought that fungi such as Trichoderma contribute largely to aerobic degradation of cellulosic biomass, our data suggested that Penicillium overwhelms them in coffee residue degradation. It was implied that polysaccharides in coffee residue are not degraded independently by different microbes, but degraded simultaneously by strains with cellulolytic, mannolytic and pectinolytic activity. Since there is no report of an ascomycete possessing all the three enzyme activities, the fast degraders are ecologically important and have the potential to be used as producers of the costly enzymes from agricultural wastes. SIGNIFICANCE AND IMPACT OF STUDY: The present results advance our understanding of microbial degradation of a phytotoxic agricultural waste, and offer a new tool for recycling it.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose

The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.     

Microbial community succession and lignocellulose degradation during agricultural waste composting

The changes of microbial community during agricultural waste composting were successfully studied by quinone profiles. Mesophilic bacteria indicated by MK-7 and mesophilic fungi containing Q-9 as major quinone were predominant and seemed to be important during the initial stage of composting. Actinobacteria indicated by a series of partially saturated and long-chain menaquinones were preponderant during the thermophilic period. While Actinobacteria, fungi and some bacteria, especially those microbes containing MK-7(H4) found in Gram-positive bacteria with a low G+C content or Actinobacteria were found cooperate during the latter maturating period. Since lignocellulsoe is abundant in the agricultural wastes and its degradation is essential for the operation of composting, it’s important to establish the correlation between the quinone profiles changes and lignocellulose degradation. The microbes containing Q-9 or Q-10(H2) as major quinone were found to be the most important hemicellulose and cellulose degrading microorganisms during composting. While the microorganisms containing Q-9(H2) as major quinone and many thermophilic Actinobacteria were believed to be responsible for lignin degradation during agricultural waste composting.     

Pectin-rich biomass as feedstock for fuel ethanol production

The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.     

Gaining electricity from in situ oxidation of hydrogen produced by fermentative cellulose degradation

AIM: To exploit the fermentative hydrogen generation and direct hydrogen oxidation for the generation of electric current from the degradation of cellulose. METHODS AND RESULTS: Utilizing the metabolic activity of the mesophilic anaerobe Clostridium cellulolyticum and the thermophilic Clostridium thermocellum we show that electricity generation is possible from cellulose fermentation. The current generation is based on an in situ oxidation of microbially synthesized hydrogen at platinum-poly(tetrafluoroaniline) (Pt-PTFA) composite electrodes. Current densities of 130 mA l(-1) (with 3 g cellulose per litre medium) were achieved in poised potential experiments under batch and semi-batch conditions. Conclusions: The presented results show that electricity generation is possible by the in situ oxidation of hydrogen, product of the anaerobic degradation of cellulose by cellulolytic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, it is shown that an insoluble complex carbohydrate like cellulose can be used for electricity generation in a microbial fuel cell. The concept represents a first step to the utilization of macromolecular biomass components for microbial electricity generation.     

Synergistic Activity of Plant Extracts with Microbial Cellulases for the Release of Free Sugars

Agricultural lignocellulosic waste such as corn stover is a potential source of inexpensive, abundant, and renewable biomass for the production of bioethanol. The enzymatic process for the economically viable breakdown of cellulose to ethanol relies on the availability of inexpensive microbial cellulases. Although the cost of cellulase has decreased in recent years, current costs still preclude the production of economically viable bioethanol from lignocellulose. Substantive efforts in this lab are being directed to transgenic production of cellulases in maize in order to boost efficiency both of production of enzymes and degradation of corn stover. We serendipitously observed that the addition of non-transgenic maize seed extracts to cellulose and microbial enzymes potentiated free sugar release by as much as 20-fold. Further, this synergistic effect between cellulase enzymes and extract was seen with a variety of plant species and tissue extracts, but varied in efficiency, and was optimal at low concentrations of cellulases. Although the nature of the synergistic molecule is not known, the use of extracts to potentiate cellulose breakdown provides opportunities for a clearer mechanistic understanding of the degradation process as well as an economical way to improve the efficiency of cellulases to produce more cost-effective bioethanol from agricultural waste.     

Use of evolutionary operation (EVOP) factorial design technique to develop a bioprocess using grease waste as a substrate for lipase production

The aim of the present work was to develop a bioprocess using EVOP-factorial design technique employing grease waste as a substrate for the production of lipase. A newly isolated fungal strain of Penicillium chrysogenum was explored for the fermentation process. Solid-state fermentation (SSF) was carried out using grease waste and Czapek-dox medium, supplemented with wheat bran. The yield of lipase was 38 U/ml when SSF was carried out at 32 °C for 8 days and grease:wheat bran:Czapek-dox media in 1:1:2 (w/w/v). Different physicochemical parameters affecting the production of lipase were optimized through evolutionary operation (EVOP) factorial design technique and after optimization yield was enhanced up to 46 U/ml at 30 °C, pH 7.0 with 1:1:2 (w/w/v) grease waste:wheat bran:Czapek-dox media. Industrial grease waste has never been reported before for the production of industrially important lipase enzyme.     

Isolation of a microbial consortium from activated sludge for the biological treatment of food waste

The bacterial community in the activated sludge of a local wastewater treatment plant was studied in an effort to understand and exploit the metabolic versatility of microorganisms for the efficient biological treatment of food waste. Microorganisms capable of and efficient in degrading domestic food waste were screened based on their ability to produce areas of clearing on selective media containing protein, fat, cellulose and starch. Nine microbial species belonging to the genera Flavobacterium, Pseudomonas, Micrococcus, Aeromonas, Xanthomonas, Vibrio and Sphingomonas were found to degrade all components of food waste. These bacteria were added to domestic wastewater and shown to cause a 60% reduction in the biochemical oxygen demand (BOD) level of wastewater compared to a control in which no microorganisms were added. The ability of the microbial consortium to degrade domestic wastewater as evidenced by the decrease in BOD levels suggests its potential for use in the biological treatment of food waste.     

Volatile fatty acids (VFAs) and methane from food waste and cow slurry: Comparison of biogas and VFA fermentation processes

The potential of various biomasses for the production of green chemicals is currently one of the key topics in the field of the circular economy. Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and they can be produced in similar reactors as biogas to increase the productivity of a digestion plant, as VFAs have more varying end uses compared to biogas and methane. In this study, the aim was to assess the biogas and VFA production of food waste (FW) and cow slurry (CS) using the anaerobic biogas plant inoculum treating the corresponding substrates. The biogas and VFA production of both biomasses were studied in identical batch scale laboratory conditions while the process performance was assessed with chemical and microbial analyses. As a result, FW and CS were shown to have different chemical performances and microbial dynamics in both VFA and biogas processes. FW as a substrate showed higher yields in both processes (435 ml CH₄/g VS_fed and 434 mg VFA/g VS_fed) due to its characteristics (pH, organic composition, microbial communities), and thus, the vast volume of CS makes it also a relevant substrate for VFA and biogas production. In this study, VFA profiles were highly dependent on the substrate and inoculum characteristics, while orders Clostridiales and Lactobacillales were connected with high VFA and butyric acid production with FW as a substrate. In conclusion, anaerobic digestion supports the implementation of the waste management hierarchy as it enables the production of renewable green chemicals from both urban and rural waste materials.     

Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85

Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains d-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove d-limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.     

餐厨废弃物资源化利用的微生物技术研究进展

简单介绍餐厨废弃物的特征和危害,综述微生物技术处理餐厨废弃物资源化的途径,如发酵提取生物降解塑料技术、厌氧发酵处理技术、微生物堆肥技术、微生物农药技术、微生物产电技术,介绍利用复合微生物菌剂降解餐厨废弃物的研究进展,分析这一新技术的发展趋势。     

烟草废弃物中的难降解有机物的微生物降解研究进展

烟草废弃物的资源化利用及无害处理过程,需要利用微生物高效降解其中的难降解物质,如木质素与尼古丁。本文主要综述烟草废弃物中难降解物质的生物降解研究进展。迄今,已经发现了不少木质素和尼古丁的微生物降解菌株,对其降解机理及应用已有不少研究报道,但其在烟草废弃物处理中的应用方面报道较少。木质素和尼古丁降解菌可以用于废次烟叶（烟梗）木质素的消减和尼古丁去除,但同时也需要考察菌株的降解能力和应用环境的适用性。具备降解木质素和尼古丁双重功能的菌株更有应用前景,但迄今发现较少。基于全基因组分析和微生物组学技术的复合菌群的研究也是重要的研究方向,将推动含木质素和尼古丁等多种难降解物质的废次烟叶的处置技术发展和实际应用。     

Eugenol stimulates lactate accumulation yet inhibits volatile fatty acid production and eliminates coliform bacteria in cattle and swine waste

AIM: To determine how eugenol affects fermentation parameters and faecal coliforms in cattle and swine waste slurries stored anaerobically. METHODS AND RESULTS: Waste slurries (faeces:urine:water, 50:35:15) were blended with and without additives and aliquoted to triplicate 1-l flasks. Faecal coliforms were eliminated in cattle and swine waste slurries within 1 or 2 days with additions of eugenol at 10.05 mm (0.15%) and 16.75 mm (0.25%). At these concentrations volatile fatty acids (VFA) were reduced ca 70 and 50% in cattle and swine waste, respectively, over 6-8 weeks. Additionally, in cattle waste, eugenol stimulated the accumulation of lactate (>180 mm) when compared with thymol treatment (20 mm lactate). In swine waste, lactate accumulation did not occur without additives; eugenol and thymol stimulated lactate accumulation to concentrations of 22 and 32 mm, respectively. CONCLUSIONS: Eugenol added to cattle waste may be more beneficial than thymol because not only does it effectively control faecal coliforms and odour (VFA production), it also stimulates lactate accumulation. This in turn, causes the pH to drop more rapidly, further inhibiting microbial activity and nutrient emissions. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant essential oils have the potential to solve some of the environmental problems associated with consolidated animal feeding operations. Thymol and eugenol reduce fermentative activity, thus, have the potential to reduce emissions of greenhouse gases and odour, and curtail transmission of pathogens in cattle and swine wastes.     

Microbial diversity in support of anaerobic biomass valorization

Microbial diversity provides an immense reservoir of functions and supports key steps in maintaining ecosystem balance through matter decomposition processes and nutrient recycling. The use of microorganisms for biomolecule production is now common, but often involves single-strain cultures. In this review, we highlight the significance of using ecosystem-derived microbial diversity for biotechnological researches. In the context of organic matter mineralization, diversity of microorganisms is essential and enhances the degradation processes. We focus on anaerobic production of biomolecules of interest from discarded biomass, which is an important issue in the context of organic waste valorization and processing. Organic waste represents an important and renewable raw material but remains underused. It is commonly accepted that anaerobic mineralization of organic waste allows the production of diverse interesting molecules within several fields of application. We provide evidence that complex and diversified microbial communities isolated from ecosystems, i.e. microbial consortia, offer considerable advantages in degrading complex organic waste, to yield biomolecules of interest. We defend our opinion that this approach is more efficient and offers enhanced potential compared to the approaches that use single strain cultures.     

Bacterial Diversity in the Hyperalkaline Allas Springs (Cyprus), a Natural Analogue for Cementitious Radioactive Waste Repository

The biogeochemical gradients that will develop across the interface between a highly alkaline cementitious geological disposal facility for intermediate level radioactive waste and the geosphere are poorly understood. In addition, there is a paucity of information about the microorganisms that may populate these environments and their role in biomineralization, gas consumption and generation, metal cycling, and on radionuclide speciation and solubility. In this study, we investigated the phylogenetic diversity of indigenous microbial communities and their potential for alkaline metal reduction in samples collected from a natural analogue for cementitious radioactive waste repositories, the hyperalkaline Allas Springs (pH up to 11.9), Troodos Mountains, Cyprus. The site is situated within an ophiolitic complex of ultrabasic rocks that are undergoing active low-temperature serpentinization, which results in hyperalkaline conditions. 16S rRNA cloning and sequencing showed that phylogenetically diverse microbial communities exist in this natural high pH environment, including Hydrogenophaga species. This indicates that alkali-tolerant hydrogen-oxidizing microorganisms could potentially colonize an alkaline geological repository, which is predicted to be rich in molecular H₂, as a result of processes including steel corrosion and cellulose biodegradation within the wastes. Moreover, microbial metal reduction was confirmed at alkaline pH in this study by enrichment microcosms and by pure cultures of bacterial isolates affiliated to the Paenibacillus and Alkaliphilus genera. Overall, these data show that a diverse range of microbiological processes can occur in high pH environments, consistent with those expected during the geodisposal of intermediate level waste. Many of these, including gas metabolism and metal reduction, have clear implications for the long-term geological disposal of radioactive waste.     

降解水稻秸秆的复合菌系及其微生物群落结构演替北大核心CSCD

【目的】比较和分析从堆肥中富集的水稻秸秆降解菌系F1和F2的纤维素分解能力、微生物群落结构及其在秸秆降解过程中的演替,从而探究微生物群落结构与秸秆降解效率的相关性。【方法】采用DNS(3,5-二硝基水杨酸,3,5-dinitrosalicylic acid)定糖法测定发酵液中的外切纤维素酶活;采用范氏(Van Soest)洗涤纤维分析法测定发酵前与发酵后的秸秆纤维素、半纤维素、木质素的含量,并计算降解率;采用16S r RNA基因序列分析和实时荧光定量PCR(Quantitative real-time PCR,Q-PCR)对秸秆降解过程中的微生物物种组成及特定的功能微生物进行定性和定量分析。【结果】复合菌系F1的水稻秸秆总降解率、纤维素降解率、半纤维素降解率显著高于复合菌系F2;2种复合菌系的外切纤维素酶活性与cel48基因的拷贝数变化趋势一致;复合菌系F1的物种较丰富,优势物种是好氧细菌,复合菌系F2的物种组成较单一,培养后期具有较高比例的厌氧纤维素分解菌;培养前4天,复合菌系F1和F2的优势物种均为Unclassified Bacillales和Bacillus;第4天之后,不同复合菌系的优势物种及丰度出现差异,F1的优势物种主要属于Bacteroidetes,F2的优势物种主要属于Firmicutes;虽然Petrimonas和Pusillimonas是培养后期的共有优势物种,但是Petrimonas在复合菌系F2中的相对丰度(38.30%)显著高于F1(9.47%),且培养第8天的F2中的Clostridiales OPB54增加至14.85%。【结论】cel48基因拷贝数变化与秸秆纤维素的降解效率、外切纤维素酶活性变化具有一定的相关性,cel48基因可作为潜在的生物分子标记监测秸秆纤维素的降解过程;微生物群落结构对秸秆纤维素的降解效率具有显著影响,Unclassified Bacillales,Bacillus,Petrimonas,Pusillimonas是复合菌系F1和F2降解秸秆纤维素过程中的重要物种。     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。     

In vitro Studies on lignocellulose degradation by microbial strains isolated from composting processes

An in vitro study of different strains isolated from composting piles in relation to their capacity to biodegrade lignocellulose was achieved. Thirteen microorganisms (five bacteria, one actinomycete, and seven fungi) isolated from compost windrows were grown on agricultural wastes and analyzed for cellulose, hemicellulose, and lignin degradation. Hemicellulose fraction was degraded to a lesser extent because only two of the isolates, B122 and B541, identified as Bacillus licheniformis and Brevibacillus parabrevis, respectively, were able to decrease the concentration of this polymer. On the contrary, most of the isolates were capable of reducing cellulose and lignin concentrations; strain B541 was the most active cellulose degrader (51%), while isolate B122 showed higher lignin degradation activity (68%). Consequently, an increase in humification indices was detected, especially with respect to humification index (HI) for both bacteria and CAH/AF in the case of strain B122. According to these data, the use of microbial inoculants as a tool to improve organic matter biodegradation processes (i.e., composting) may become important if microorganisms’ capabilities are in accordance with the final characteristics required in the product (high humic content, lignin content decrease, cellulose concentration decrease, etc.).     

Ethanol production from candidate energy crops: water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.)

Fermentation modes and microorganisms related to two typical free-floating aquatic plants, water hyacinth and water lettuce, were investigated for their use in ethanol production. Except for arabinose, sugar contents in water lettuce resembled those in water hyacinth leaves. Water lettuce had slightly higher starch contents and lower contents of cellulose and hemicellulose. A traditional strain, Saccharomyces cerevisiae NBRC 2346, produced 14.4 and 14.9 g l(-1) ethanol, respectively, from water hyacinth and water lettuce. Moreover, a recombinant strain, Escherichia coli KO11, produced 16.9 and 16.2 g l(-1) ethanol in the simultaneous saccharification and fermentation mode (SSF), which was more effective than the separated hydrolysis and fermentation mode (SHF). The ethanol yield per unit biomass was comparable to those reported for other agricultural biomasses: 0.14-0.17 g g-dry(-1) for water hyacinth and 0.15-0.16 g g-dry(-1) for water lettuce.