Roberto Oberti

Two-Stage vs Single-Stage Thermophilic Anaerobic Digestion: Comparison of Energy Production and Biodegradation Efficiencies

Two-stage anaerobic digestion (AD) for integrated biohydrogen and biomethane production from organic materials has been reported to promise higher process efficiency and energy recoveries as compared to traditional one-stage AD. This work presents a comparison between two-stage (reactors R1 and R2) and one-stage (reactor R3) AD systems, fed with identical organic substrates and loading rates, focusing the attention on chemical and microbiological aspects. Contrary to previous experiences, no significant differences in overall energy recovery were found for the two-stage and one-stage AD systems. However, an accumulation in R2 of undegraded intermediate metabolites (volatile fatty acids, ketones, amines, amino acids, and phenols) was observed by GC-MS. These compounds were thought to be both cause and effect of this partial inefficiency of the two-stage system, as confirmed also by the less diverse, and thereby less efficient, population of fermentative bacteria observed (by PCR-DGGE) in R2. The extreme environment of R1 (low pH and high metabolites concentrations) probably acted as selector of metabolic pathways, favoring H(2)-producing bacteria able to degrade such a wide variability of intermediate metabolites while limiting other strains. Therefore, if two-stage AD may potentially lead to higher energy recoveries, further efforts should be directed to ensure process efficiency and stability.

Biohydrogen from thermophilic co-fermentation of swine manure with fruit and vegetable waste: Maximizing stable production without pH control

Hydrogen production by dark fermentation may suffer of inhibition or instability due to pH deviations from optimality. The co-fermentation of promptly degradable feedstock with alkali-rich materials, such as livestock wastes, may represent a feasible and easy to implement approach to avoid external adjustments of pH. Experiments were designed to investigate the effect of the mixing ratio of fruit-vegetable waste with swine manure with the aim of maximizing biohydrogen production while obtaining process stability through the endogenous alkalinity of manure. Fruit-vegetable/swine manure ratio of 35/65 and HRT of 2d resulted to give the highest production rate of 3.27 ± 0.51 L(H2)L(-1)d(-1), with a corresponding hydrogen yield of 126 ± 22 mL(H2)g(-1)(VS-added) and H2 content in the biogas of 42 ± 5%. At these operating conditions the process exhibited also one of the highest measured stability, with daily productions deviating for less than 14% from the average.

Microbial community structure and dynamics in two-stage vs single-stage thermophilic anaerobic digestion of mixed swine slurry and market bio-waste.

The microbial community of a thermophilic two-stage process was monitored during two-months operation and compared to a conventional single-stage process. Qualitative and quantitative microbial dynamics were analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and real-time PCR techniques, respectively. The bacterial community was dominated by heat-shock resistant, spore-forming clostridia in the two-stage process, whereas a more diverse and dynamic community (Firmicutes, Bacteroidetes, Synergistes) was observed in the single-stage process. A significant evolution of bacterial community occurred over time in the acidogenic phase of the two-phase process with the selection of few dominant species associated to stable hydrogen production. The archaeal community, dominated by the acetoclastic Methanosarcinales in both methanogen reactors, showed a significant diversity change in the single-stage process after a period of adaptation to the feeding conditions, compared to a constant stability in the methanogenic reactor of the two-stage process. The more diverse and dynamic bacterial and archaeal community of single-stage process compared to the two-stage process accounted for the best degradation activity, and consequently the best performance, in this reactor. The microbiological perspective proved a useful tool for a better understanding and comparison of anaerobic digestion processes.

Testing of a simplified LED based vis/NIR system for rapid ripeness evaluation of white grape (Vitis vinifera L.) for Franciacorta wine

The aim of this work was to test a simplified optical prototype for a rapid estimation of the ripening parameters of white grape for Franciacorta wine directly in field. Spectral acquisition based on reflectance at four wavelengths (630, 690, 750 and 850 nm) was proposed. The integration of a simple processing algorithm in the microcontroller software would allow to visualize real time values of spectral reflectance. Non-destructive analyses were carried out on 95 grape bunches for a total of 475 berries. Samplings were performed weekly during the last ripening stages. Optical measurements were carried out both using the simplified system and a portable commercial vis/NIR spectrophotometer, as reference instrument for performance comparison. Chemometric analyses were performed in order to extract the maximum useful information from optical data. Principal component analysis (PCA) was performed for a preliminary evaluation of the data. Correlations between the optical data matrix and ripening parameters (total soluble solids content, SSC; titratable acidity, TA) were carried out using partial least square (PLS) regression for spectra and using multiple linear regression (MLR) for data from the simplified device. Classification analysis were also performed with the aim of discriminate ripe and unripe samples. PCA, MLR and classification analyses show the effectiveness of the simplified system in separating samples among different sampling dates and in discriminating ripe from unripe samples. Finally, simple equations for SSC and TA prediction were calculated.

OPTICAL TECHNIQUES TO ESTIMATE THE RIPENESS OF RED-PIGMENTED FRUITS

During fruit ripening, chlorophyll degradation is responsible for the degreening of the ground color, which is a well-established ripeness indicator for several species. In completely red-pigmented cultivars of fruits such as apples and peaches, this process is not visible, being masked by anthocyanins in the skin. Two different optical systems were developed to non-destructively assess the chlorophyll content in these fruits, to estimate ripeness, and to optimize harvesting and postharvest management. A fluorescence imaging system equipped with a UV-blue actinic light was used to obtain fluorescence images of fruit in which the gray level of pixels correlated (R2 = 0.81) with the firmness of fresh apples (Malus domestica cv. Red Delicious). With this technique it was possible to estimate changes in the firmness and soluble solids sugar content of stored Red Delicious apples undergoing no detectable hue change in the skin. Using the same system with a red actinic light, fluorescence correlated fairly well with firmness for fresh peaches and nectarines (Prunus persica cv. Elegant Lady, Summer Rich, and Morsiani 90), even though the detected fluorescence signal was low in intensity. A laser-diode based, dual-band reflectance probe was developed and tested on fresh peaches (cv. Summer Rich) and stored apples (cv. Royal Gala). The R/IR index, defined as the ratio of the signal measured in red and near-infrared bands, was found to correlate with the chlorophyll content of the fruits (R2 = 0.66), regardless of fruit species and anthocyanin presence. The R/IR index was used to track the postharvest ripening process for fresh peaches harvested at different maturity stages.

Evaluation of borage extracts as potential biostimulant using a phenomic, agronomic, physiological and biochemical approach

Biostimulants are substances able to improve water and nutrient use efficiency and counteract stress factors by enhancing primary and secondary metabolism. Premise of the work was to exploit raw extracts from leaves (LE) or flowers (FE) of Borago officinalis L., to enhance yield and quality of Lactuca sativa ‘Longifolia,’ and to set up a protocol to assess their effects. To this aim, an integrated study on agronomic, physiological and biochemical aspects, including also a phenomic approach, has been adopted. Extracts were diluted to 1 or 10 mL L–1, sprayed onto lettuce plants at the middle of the growing cycle and 1 day before harvest. Control plants were treated with water. Non-destructive analyses were conducted to assess the effect of extracts on biomass with an innovative imaging technique, and on leaf photosynthetic efficiency (chlorophyll a fluorescence and leaf gas exchanges). At harvest, the levels of ethylene, photosynthetic pigments, nitrate, and primary (sucrose and total sugars) and secondary (total phenols and flavonoids) metabolites, including the activity and levels of phenylalanine ammonia lyase (PAL) were assessed. Moreover, a preliminary study of the effects during postharvest was performed. Borage extracts enhanced the primary metabolism by increasing leaf pigments and photosynthetic activity. Plant fresh weight increased upon treatments with 10 mL L–1 doses, as correctly estimated by multi-view angles images. Chlorophyll a fluorescence data showed that FEs were able to increase the number of active reaction centers per cross section; a similar trend was observed for the performance index. Ethylene was three-fold lower in FEs treatments. Nitrate and sugar levels did not change in response to the different treatments. Total flavonoids and phenols, as well as the total protein levels, the in vitro PAL specific activity, and the levels of PAL-like polypeptides were increased by all borage extracts, with particular regard to FEs. FEs also proved efficient in preventing degradation and inducing an increase in photosynthetic pigments during storage. In conclusion, borage extracts, with particular regard to the flower ones, appear to indeed exert biostimulant effects on lettuce; future work will be required to further investigate on their efficacy in different conditions and/or species.

Detection of Fungal Diseases Optically and Pathogen Inoculum by Air Sampling

Practical solutions to measure temporal and spatial differences in the epidemics of specific fungal plant diseases are described here. For diseases that develop from widespread airborne inoculum , timing of disease control methods are key. Air sampling , integrated with appropriate diagnostic methods can be used to identify and quantify the presence of pathogen inoculum in order to guide spray decisions. Where diseases are already established but with spatially variable severity (disease foci ), spatially selective spraying of crops is possible using different optical disease detection methods and knowledge of pathogen biology to estimate an area of latent (invisible but developing) infection around disease foci. Spatially-selective spraying mediated by optical sensors may also be beneficial when there are crop patches that have low yield potential due to other factors such as poor emergence, moisture or nutrient stress, or soil compaction. Precision agriculture methods to improve the efficiency of fungicide applications in terms of timing and selective spatial application can optimise the use of fungicides in integrated crop production systems to provide the lowest environmental impact per unit of produce while maintaining a high protection efficacy.

Chlorophyll fluorescence sensing for early detection of crop’s diseases symptoms

A chlorophyll fluorescence imaging system based on a filtered xenon lamp, providing actinic light in UV and violet bands, and on a high resolution camera equipped with a 690nm (FWHM=10nm) passband filter, for single band measurements, and with a four bands beam splitter with pass-band filters (450nm, 550nm, 690nm, 740nm, all with a FWHM=10nm), for multispectral measurements, was implemented and applied on wheat plants inoculated with different fungal infections, with the aim of investigating the potential of such a technique for detecting plants disease symptoms. In steady state fluorescence images of attached leaves acquired at 690nm in laboratory conditions, the symptoms appear as highly emitting spots at sub-millimetric or millimetric scale which, with the progress of the disease, develop in larger, low emitting lesions surrounded by high intensity halo. Even if the changes in emission pattern are limited to the neighborhoods of the infection point; this technique allowed to detect disease presence before visible symptoms appear. Kinetic fluorescence imaging performed by acquiring a sequence of images at 690nm during an actinic illumination period of several minutes, allowed to find differences between diseased and healthy areas, even at very early stages, both in terms of intensity and time-dependence of emission. Nevertheless, the excitation/sensing period of several minutes on which this technique is based, limits practical field applications on moving vehicles. Multispectral fluorescence imaging in field conditions resulted unsuccessful during day-time measurements due to plants saturation by long-exposure to direct sunlight and to the interference of the diffuse environmental illumination. On the contrary, night-time imaging confirmed the high potential of this technique for disease detection and quantifications. In particular, an image analysis algorithm based on the ratio of fluorescence images at 550nm and 690nm was implemented, allowing to discriminate plants lesions and to map the disease severity in experimental plots in agreement with visual inspection made by a pathologist.

A Simplified, Light Emitting Diode (LED) Based, Modular System to be Used for the Rapid Evaluation of Fruit and Vegetable Quality: Development and Validation on Dye Solutions.

NIR spectroscopy has proven to be one of the most efficient and ready to transfer tools to monitor product’s quality. Portable VIS/NIR instruments are particularly versatile and suitable for field use to monitor the ripening process or quality parameters. The aim of this work is to develop and evaluate a new simplified optoelectronic system for potential measurements on fruit and vegetables directly in the field. The development, characterization and validation of an operative prototype is discussed. LED technology was chosen for the design, and spectral acquisition at four specific wavelengths (630, 690, 750 and 850 nm) was proposed. Nevertheless, attention was given to the modularity and versatility of the system. Indeed, the possibility to change the light sources module with other wavelengths allows one to adapt the use of the same device for different foreseeable applications and objectives, e.g., ripeness evaluation, detection of particular diseases and disorders, chemical and physical property prediction, shelf life analysis, as well as for different natures of products (berry, leaf or liquid). Validation tests on blue dye water solutions have shown the capability of the system of discriminating low levels of reflectance, with a repeatability characterized by a standard deviation proportional to the measured intensity and in general limited to 2%–4%.

Looking for practical tools to achieve next-future applicability of dark fermentation to produce bio-hydrogen from organic materials in Continuously Stirred Tank Reactors

This study aimed at finding applicable tools for favouring dark fermentation application in full-scale biogas plants in the next future. Firstly, the focus was obtaining mixed microbial cultures from natural sources (soil-inocula and anaerobically digested materials), able to efficiently produce bio-hydrogen by dark fermentation. Batch reactors with proper substrate (1 gL(glucose)(-1)) and metabolites concentrations, allowed high H(2) yields (2.8 ± 0.66 mol H(2)mol(glucose)(-1)), comparable to pure microbial cultures achievements. The application of this methodology to four organic substrates, of possible interest for full-scale plants, showed promising and repeatable bio-H(2) potential (BHP=202 ± 3 NL(H2)kg(VS)(-1)) from organic fraction of municipal source-separated waste (OFMSW). Nevertheless, the fermentation in a lab-scale CSTR (nowadays the most diffused typology of biogas-plant) of a concentrated organic mixture of OFMSW (126 g(TS)L(-1)) resulted in only 30% of its BHP, showing that further improvements are still needed for future full-scale applications of dark fermentation.