INTRODUCTION

The processing of fruit and vegetables for making juices and wines generates large amounts of vegetable residues (pomace) which are landfilled or fed to animals. Biowaste (the biodegradable fraction of municipal household waste collected separately) can have a similar composition - mainly vegetable scraps. The purpose of this project is to look at biological methods (fermentations) of adding value to such materials which at present are generally fed to animals or discarded in landfills.

SUMMARY

This Item covers the work completed in the first year of this project which aims to increase the value of various agro-food processing wastes through fermentation. At present large amounts of vegetable wastes (pomace) are generated during juice extraction from fruit and vegetables. These are often dumped in landfills or fed to animals. The organic fraction of municipal waste (here termed biowaste) may have a similar composition to pomace and may also be upgraded in this way. The project considers a number of different wastes (carrot, potato, grape) and a number of products (dietary fibre, enzymes, waste water processing additive, colours and flavours). The main conversion process used for upgrading under investigation is bacterial fermentation using either lactobacilli or bacilli.

OBJECTIVE

The processing of fruit and vegetables for making juices and wines leads to large amounts of vegetable residues (pomace), which are landfilled or fed to animals. Biowaste, another type of residue is the biodegradable fraction of municipal household waste and is often collected separately.

The high crude fibre content of vegetable pomace suggests its utilization as a dietary fibre bread improver. An enrichment of different products with crude fibre compounds can raise the dietary fibre uptake of the population. Processing of the vegetable residues to a dietary fibre food additive can be done by lactic acid fermentation, leading to a transformation of low molecular materials and to a microbial stabilization.

Potato waste can be used as a substrate for the low-cost microbial production of enzymes like alpha-amylase, widely used in the food industry and the textile industry. After enzyme extraction, a pomace remains that can be used for the production of technical-grade lactic acid.

Grape waste can be used for the production of wine-pip-oil, colourings and grape flavour. All of the three processes can be performed in a much more efficient way if enzymatic treatment steps are involved. The remaining pomace can be processed to lactic acid. Bioresidue products with too low an overall quality cannot be upgraded to food grade products and will be used for low-cost production of lactic acid for use as a floc-forming supplement in wastewater treatment. Indeed, the loading capacity of a biological aerobic or anaerobic wastewater treatment system is essentially determined by the amount of active biomass retained in the reactor, which can be positively influenced upon by lactic acid. An open system fermentation is to be developed in which bioresidue products are used as substrates for low-cost lactic acid production. The effect of this feed-grade lactic acid on sludge settling properties in a number of different wastewaters and in the different reactor types used for wastewater purification is to be studied.

ACTIVITIES

Conversion of vegetable residues to a dietary fibre food additive was investigated using lactic acid fermentation. The fermentation process should transform low molecular materials leading to a fibre product which is biologically stable. The possibility of using potato waste as a substrate for the production of microbial amylase and technical grade lactic acid was established. Grape waste has been investigated as a source of wine pip oil, colorings and grape flavour. It has been shown that these processes can be enhanced by incorporating enzymatic treatment steps. It has also been shown that low quality substrates (wastes) can be used for low-cost production of lactic acid for use as a floc-forming supplement in wastewater treatment.

Good lactic acid fermentation of carrot pomace was observed, using a lactobacillus isolated from sour dough at ambient temperatures in stainless steel vessels on the production site. Usually the fermented pomace can be kept for a week without deterioration. However, if the carrots have been previously stored and the temperature is low (around 15°C), the activity of the lactic acid bacteria is sub-optimal and mould growth can occur. Following spray drying and milling, the fibre has been used in baking trials, with mixed results on bread volume as well as crumb and crust structure. Addition of water gave some improvement.

In studies of grape pomace fermentation there was found to be a progressive, temperature inactivation of both yeasts and lactic acid bacteria, leading to the selection of a number of resistant strains. Most yeasts have been identified and a number of bacteria have been isolated but not yet identified.

Work on enzyme assisted extraction of pip oil and colour were found to be of less value than expected since this is an alternative methods of extraction and preparation of existing products which have markets which are themselves limited and to a large extent satisfied.

The use of carrot pomace in water treatment focused on active and dense granular sludge in upflow anaerobic sludge blanket (UASB) reactors, which depends on the rapidly acidifying properties (RACOD) of the waste water and the surface tension of the reactor liquid. Several experiments were carried out with lab scale UASB reactors in order to develop a kind of granular growth supplement (GGS) which enhances the sludge granulation and the granular sludge bed stability. Best results were obtained with a GGS containing RACOD in the form of a sucrose/starch mixture and linear alkylbenzene sulphonate (LABS). By adding 20% of RACOD on top of the original influent COD and adjusting the reactor liquid surface tension, granular growth and sludge bed stability could be enhanced significantly within 40 days. When carrot pulp/pomace was applied as an alternative RACOD source under various conditions. The best results were obtained when fresh carrot pulp was added to the lab scale UASB reactors in an integrated recycle contact chamber. This was tried out on a real industrial waste water and was shown to be successful.

Enzyme production on potato waste has yielded two a-amylases from B. stearothermophilus: one is cell bound, the other one is extracellular. These have different characteristics with a relatively low pH optimum which is an advantage in the first step of the starch liquefaction process: the formation of maltodextrins a temperatures of 110°C and at pH 6 7. Addition of extra medium compounds is not necessary; pure potato waste is sufficient for the fermentation. A number of structural features that could be responsible for increased thermostability of the enzymes were identified by X ray diffraction analysis. Five or six salt bridges, new hydrophobic interactions and new hydrogen bonds, absent from the thermolabile CGTase structures, were identified.

FUTURE WORK

An emphasis is placed on further optimisation of the amylase production by cloning and expression of the gene in a more suitable host (B. subtilis).

PARTICIPANTS

Institute of Food Technology, University of Bonn (Germany), EDEN Waren, Hunfeld (Germany), Laboratory for Microbial Ecology, University of Gent (Belgium), ATO DLO, Wageningen (Netherlands), Escola Superior de Bio-tecnologia, Porto (Portugal).

Second Project Progress Report Summary

Introduction
This report covers that part of the project on fermentative utilization of biowaste as a source of raw material for anaerobic digestion. In the work reported here the emphasis was on the use of carrot pulp/pomace as an alternative source of rapidly acidifying COD (RACOD). The main task is to improve the sludge granulation process in UASB reactors by the application of carrot waste derived products. In a broader sense, the production of novel types of products, applicable in waste water treatment that enhance process stability and/or curing of problematic reactor performances, is envisaged.

Upflow Anaerobic Sludge Blanket (UASB) wastewater (pre-) treatment systems represent a proven technology for a wide range of very different industrial effluents. including those containing toxic and/or inhibitory compounds. The process can also be used for treatment of domestic wastewater with temperatures around 15 degrees centigrade. The anaerobic sludge blanket reactor concept is based on the inherent settling properties of the sludge that occurs, provided the sludge is not exposed to high rates of mechanical agitation. For this reason in UASB reactors mechanical mixing is either omitted completely or applied at a relatively low intensity and/or intermittently. To achieving the required contact between sludge and wastewater, the conventional UASB system relies on the agitation brought about by bubbling of the biogas produced together with an even distribution of the influent feed stream at the bottom of the reactor. Sometimes the hydraulic upflow velocity is increased by applying effluent recycling. Under these conditions sludge aggregates may be dispersed. Generally the dispersed sludge aggregates can be retained in the reactor by separating the biogas using a gas collector assembly, is placed in the upper part of the reactor where biogas is released and sludge particles can coalesce and settle, falling back into the digester compartment

The wash-out of floating sludge particles can, in general, be effectively prevented for medium and relatively low strength wastewater by installing a baffle in front of the effluent weir. Where very high sludge loading rates are imposed to the system, the presence of gas within the sludge aggregates may cause floatation of the sludge particles. In this case, specific designs of the gas solids separator may become necessary. Some commercial, full-scale UASB reactors, employ sophisticated multistep gas separators.

Start-up with granular sludge. Large amounts of high quality (granular) excess sludge are becoming more available. Since this represents an almost ideal seed material, the start-up of new installations can be completed within a few days. This is true even for wastewaters that are completely different from the wastewater originally being treated. The granule formation is believed to be mediated by an autoimmobilisation processes, although factors governing granulation are still unclear. Our results showed that for good granulation and stable UASB reactor performance the overall reactor conditions should be conducive to the growth of granules in which acidogens are predominant at the outer layer of the granules. The presence of RACOD in the feed at 10 to 20% of the total COD content, combined with a low liquid surface tension promotes layered granules.

The use of carrot pulp as a source of RACOD source was investigated and reported on previously when a sludge floatation problem induced by the carrot pulp fibres when present in the UASB reactors was noted. This problem was solved by adding the fresh carrot pulp in an integrated liquefying chamber so that the hydrolysis of the carrot pulp and the acidogenesis of the produced RACOD were separated spatially. However, before evaluation in practice could occur, a more practical method of adding the carrot pulp had to be found. Therefore alternative methods were investigated during year three of the project.

Activities
So far the best results have been obtained by adding fresh carrot pulp in an integrated liquefying chamber in a recycle loop. In this way the hydrolysis of the carrot pulp and the acidogenesis of the solubilised material (RACOD) were separated spatially. However, before the system could be evaluated in detail a more practical way of adding the carrot pulp had to be found. Therefore. several alternatives were investigated. In the first option an integrated bag filter was used in which hydrolysis of the carrot pulp took place in such a way that the residual carrot fibres were prevented from entering the UASB reactor. Due to the accumulation of microbial biomass in the filter, hydrolysis and acidification took place at the same time, with very little or no enhancement of sludge granulation. Therefore, the experimental set-up was modified for a second experiment in which the hydrolysis of the carrot pulp was carried out batchwise in a separate bag filter. The hydrolysate was then used as influent for the UASB reactor after the addition of VFA (Volatile Fatty Acids ) and nutrients. This modification resulted in only a partial acidification with an average RACOD yield of 53 plus or minus 11 percent of the total COD. When using the hydrolysate as feed for the UASB reactor. the granular yield was increased by 35 percent. The general reactor parameters such as COD removal efficiency and the pH were not significantly influenced. However, the accumulation of residual, fibrous carrot pulp in the bag filter was a serious drawback of this option, since it would require a laborious sequence of treatments to dewater and stabilise the residual pulp. This conclusion was based on the results of laboratory scale experiments carried out on dewatering of carrot pomace.

Another approach was the direct treatment of the carrot pomace together with the waste water originating from the carrot processing company Pinguin Inc. (Langemark, Belgium) in a full scale UASB reactor. In spite of the presence of suspended solids that showed poor settleability. This was especially true of the fibrous carrot pomace in the influent. However, no serious sludge floatation was observed in the UASB reactor, provided the advanced floatation unit within the system, was working properly and the actual COD load was significantly lower than the design load (10.68 kg COD per cubic metre per day). Therefore, it was decided to adopt this approach of using the carrot pulp as such, without removal of the fibrous fraction.

In further work, efforts were focused on the enzymatic liquefaction of the carrot pulp in order to minimise the fibre content and to obtain a solution with a high ratio of soluble to total COD. Various mixtures of pectinolytic and cellulolytic enzymes were tried out in order to reduce the Total Dietary Fibre (TDF) content of the carrot pulp. The best results were obtained using Pectinex ultra SP-L at pH 5, after addition of water to reduce the DM (Dry Matter) content to about 15%. However, this took prolonged incubation over more than 6 hours. Furthermore, pressing was still required in order to obtain a solution with a high soluble COD. The resulting liquid phase had a soluble COD content of approximately 110 kg. per cubic meter, and therefore could be used as a liquid source of RACOD. However, the amount of enzyme required to produce this solution was too high to make this procedure economically viable. The seasonal production of the carrot pulp waste product makes the use of a liquid and stable form of Granular Growth Supplement (GGS) more feasible in practice . This approach merits further investigation, especially in case low cost enzyme sources become available.