Naar veilig en efficiënt gebruik van insectenfrass in duurzame landbouw - Microbiologische kwaliteit en plantengroei-bevorderende effecten op de ontwikkeling van de bonenplant

The growing world population challenges agriculture to produce sufficient food, while also shifting to more sustainable production methods. How insect farming has emerged as a small but promising contribution to the solutions is covered in Chapters 1 and 2. Through low-environmental impact production processes and efficient conversion of low-value food waste streams into high-value protein for both animal feed/human food, insect farming already ticks some of the sustainability and circularity boxes. Efficient valorisation of the residual rearing substrate (frass) as fertiliser/biostimulant in crop production would further augment the biocircularity of the insect production chain. However, several obstacles remain due to knowledge gaps about the effectiveness of frass addition and the nature of the frass growth-promoting properties. Knowledge regarding the impact of the heat treatment, imposed by EU regulations to mitigate the spread of potential food pathogens, on the frass properties is also limited. In Chapter 3, the impact of the reference heat treatment at 70 °C for 1 h and the effect of treatments at lower temperatures and/or shorter times on the intrinsic properties and the microbiological quality/safety of black soldier fly larvae (Hermetia illucens, HI) and yellow mealworm (Tenebrio molitor, TM) frass are explored, next to microbial dynamics during frass storage. The results indicated that untreated TM frass, dry and low in water activity, was more microbiologically safe/stable than untreated HI frass, high in moisture content and water activity. Enterobacteriaceae numbers (including E. coli) were higher in HI frass, Enterococcaceae counts were similar in HI and TM frass and Salmonella was absent in both. Reference heat treatment reduced the total bacterial load slightly. In HI frass, Enterobacteriaceae were reduced to counts below 1 log cfu/g, inoculated Salmonella spp. were completely eradicated and Enterococcaceae counts were reduced below the legal limit of 3 log cfu/g. For TM frass, reduction to counts below 3 log cfu/g was not always achieved and Salmonella spp. were not always eliminated. Treatments at 60 °C for at least 15 minutes secured HI frass safety, while for TM frass minimally 70 °C was required. Refrigerated or ambient temperature storage up to two weeks did not increase microbial counts. Heat treatment in an industrial context differs from the settings applied in the Chapter 3 experiments. Therefore, Chapter 7 investigates pilot-scale hygienisation treatments. Oven drying is compared to alternatives, such as pelleting and composting, concerning both effectiveness and practical applicability. Oven drying generated microbiologically safe HI and TM frass, but the process is expensive, and milder treatment could achieve a similar outcome. Pelleting was found suited for TM frass hygienisation, given its low moisture content and low E. coli counts. HI frass required a drying step prior to pelleting, while the pelleting process itself did not change its legal conformity. Composting of HI frass generated a safe product on condition that the process temperatures reached induced sufficient E. coli reduction. Composting was found less suited for TM frass hygienisation. Whether or not the applied treatments generated frass complying with the EU standards on microbiological safety depended on the number of E. coli. Most often present in smaller numbers compared to Enterococcaceae, the species is also more heat-sensitive and therefore more easily reduced. The legally imposed heat treatment may sometimes be unnecessary for TM frass and may often be too strict for both HI and TM frass. Milder heat treatments or alternative processing may also generate safe products and at the same time lower the impact on the frass beneficial microbial community. The valorisation potential of HI and TM frass in crop cultivation is explored in Chapters 4, 5 and 7 of this PhD dissertation. The research outcomes of Chapter 4 showed a generally positive stimulus on green bean growth and development, resulting from 2 % HI and TM frass application, both untreated and thermally dried. Seed germination was not restricted and frass-amended plants continued to grow until harvest while growth of unamended plants slowed or stagnated. Frass amendment resulted in plants with higher leaf chlorophyll contents, increased fresh plant biomass and pod yield. In addition, the mineral nutrient and protein content of bean plant leaves and pods also increased. These beneficial influences were related to improved light use efficiency in the bean leaves but could only partially be attributed to enhanced nutrient supply from the 2 % frass. Factors behind the observed positive stimuli were further investigated in Chapter 5. Differences between the effects of 0.5 to 4 % frass application rates were minor. Mineralisation experiments showed that upon addition of 2 % untreated and thermally dried HI frass to potting soil, about 20 % of the frass total nitrogen became available for plant uptake. Addition of 2 % HI frass improved bean plant development similarly or even better than addition of inorganic NPK fertiliser, supplying comparable amounts of available N. These findings underscored that besides its nutrient effect, frass also stimulates plant growth through biostimulant action, probably related to its microbial community. Pelletised HI and TM frass exerted a beneficial influence on bean development, as was found for amendment with untreated and thermally dried frass. The findings in Chapters 4 and 7 further showed that at similar nutrient supply, the impacts seemed less outspoken when the frass was applied after thermal oven drying or pelleting compared to untreated. This pointed at an influence of the hygienisation process, more severe for oven drying than for pelleting, on the frass plant growth-stimulating properties, potentially linked to the survival rate of potential beneficial microorganisms. In Chapter 6, untreated and thermally dried Hermetia illucens frass proved sources of microorganisms with ability to thrive in the plant rhizosphere and exert stimulating effects on plant development, such as enhancement of nutrient availability (produce ammonia and/or solubilise phosphate) and production of phytohormones. Some promising selected isolates improved Arabidopsis thaliana root development (shorter root/higher root hair density). Sequencing results showed that isolates belonged to the genera Providencia, Proteus, Corynebacterium, Bacillus, Heyndrickxia and Paenibacillus. Several species of these genera were associated with the Hermetia illucens larvae gut core microbiota and known to possess plant growth-promoting properties. The hygienisation treatment, aimed at reduction of potential human pathogens, also reduced or eliminated several of the promising HI frass bacteria, mainly heat-tolerant/spore-forming taxa remained. All these outcomes confirmed that application of frass not only provided plant nutrients but also exerted additional biostimulant effects, which may however be compromised by frass processing. In the final chapter of this PhD dissertation, the insights gained, and conclusions drawn, are used in recommendations to policy makers and insect producers, regarding the microbiological safety and processing of insect frass, aimed at augmenting its valorisation potential in sustainable agriculture. The suggestions for future research may help to unravel the promising but still largely unknown properties of insect frass.