Industrial Algae Measurements

Transcription

Industrial Algae Measurements
October 2013 | Version 6.0
A publication of ABO’s
Technical Standards Committee
through funding from the Algae Foundation
© 2014 Algae Biomass Organization
About the Algae Biomass Organization
Founded in 2008, the Algae Biomass Organization (ABO) is a non-profit organization whose mission is to promote the development of viable
commercial markets for renewable and sustainable products derived from algae. Our membership is comprised of people, companies, and
organizations across the value chain. More information about the ABO, including membership, costs, benefits, members and their affiliations, is
available at our website: www.algaebiomass.org.
The Technical Standards Committee is dedicated to the following functions:
•
•
•
•
Developing and advocating algal industry standards and best practices
Liaising with ABO members, other standards organizations and government
Facilitating information flow between industry stakeholders
Reviewing ABO technical positions and recommendations
About the IAM 6.0 Document
This Guidance Document recommends minimum descriptive parameters and measurement methodologies required to fully characterize the
economic and environmental inputs and outputs of an aquatic biomass operation. Voluntary adoption of uniform language and methodologies will
accelerate industry growth and unify research.
The Industrial Algae Measurements 6.0 recommendations contained within this document are intended to be useful in characterizing a
broad range of aquatic production operations. The Algae Biomass Organization (ABO) is primarily focused on the production of eukaryotic algae
(both macro and micro) and cyanobacteria (blue-green algae). However, the cultivation of alternative aquatic crops such as duckweed or other
photosynthetic organisms will also benefit from the information provided in this document because many of the environmental inputs and outputs
are common. The language and methodology recommendations in this document will also equally serve heterotrophic algal cultivation, algalbacterial binary systems and multi-trophic systems that employ higher organisms to harvest or otherwise enhance value.
Document Revision:
For more information, please see: http://www.algaebiomass.org
This Minimum Descriptive Language document will be periodically revised based on ongoing stakeholder input.
Staff
Committees
Executive Director – Mary Rosenthal
General Counsel – Andrew Braff, Wilson Sonsini Goodrich & Rosati
Administrative Coordinator - Barb Scheevel
Administrative Assistant – Nancy Byrne
Website Manager – Riley Dempsey
Events Committee
Chair – Philip Pienkos, National Renewable Energy Laboratory
Board of Directors
Chair – Margaret McCormick, Matrix Genetics
Vice Chair – Tim Burns, Bioprocess Algae LLC
Secretary – Tom Byrne, Algaedyne Corporation
Mark Allen, Accelergy Corporation
John Benemann, MicroBio Engineering, Inc.
Julie Felgar, The Boeing Company
David Hazlebeck, General Atomics
Greg Mitchell, Scripps Institution of Oceanography
Joel Murdock, FedEx Express
Jose Olivares, Los Alamos National Laboratory
Philip Pienkos, National Renewal Energy Laboratory
James Rekoske, UOP/Honeywell
Todd Taylor, Fredrikson & Byron Law, P.A.
Paul Woods, Algenol Biofuels, Inc.
Tim Zenk, Sapphire Energy
Member Development Committee
Chair – Todd Taylor, Fredrikson & Byron Law, P.A.
Vice Chair – John Benemann, MicroBio Engineering, Inc.
Bylaw & Governance Committee
Chair - Mark Allen, Accelergy Corporation
Algae Biomass Organization | 1
To comment on this document or recommend improvements, please send suggestions to Barb Scheevel, Administrative Coordinator, via e-mail
at bscheevel@algaebiomass.org.
For further information on this document and the ABO’s Technical Standards Committee, please contact Dr. Lieve Laurens, Chair of the Technical
Standards Committee via e-mail at Lieve.Laurens@nrel.gov or via phone at (303) 384-6196.
ABO Technical Standards Committee Authors:
Technical Standards Committee
Chair – Lieve Laurens, National Renewable Energy Laboratory
Co-Chair – Keith Cooksey, Environmental Biotechnology Consultants
Dr. Lieve Laurens - Committee Chair, Algal Research Scientist, National Renewable Energy Laboratory
Director Recruitment Committee
Chair – Tim Burns, BioProcess Algae, LLC
Jim Sears, New Product Development, Boulder Labs Inc., Former ABO Committee Chair and CTO, A2BE Carbon Capture LLC
Peer Review Commitee
Co-Chair – Keith Cooksey, Environmental Biotechnology Consultants
Co-Chair – John Benemann, MicroBio Engineering, Inc.
Dr. Mark Edwards, Professor, Arizona State University Morrison School of Agribusiness and Resource Management, Vice President at Algae
Biosciences Inc
Executive Policy Council
Chair – Tim Zenk, Sapphire Energy
Dr. Keith Cooksey - Committee Co-Chair, Environmental Biotechnology Consultants, Professor Emeritus, Montana State University,
ABO Board Member
Dr. Rose Ann Cattolico, Professor of Algal Biology, University of Washington
Steve Howell, President and founder of MARC-IV, Chairman of the ASTM task force on biodiesel standards
Adonis Neblett, Attorney, Minnesota Pollution Control Agency
Dr. Robert McCormick, Principal Engineer, Fuels Performance, National Renewable Energy Laboratory
Dr. Philip Pienkos, Applied Sciences Group Manager, National Renewable Energy Laboratory
Gina Clapper, Technical Specialists, AOCS American Oil Chemists Society
Pat Ahlm, Assistant Director, Government and Regulatory Affairs at Algenol Biofuels Inc
Table of Contents
About the IAM 6.0 Document
Executive Summary
The Challenge and ABO’s Green Box Solution
Industrial Measurement of Large Scale Operations
Appendix A: Measuring Dry Weight and Compositional Analysis of Algal Biomass
Appendix B: Basics of Lifecycle and Techno-Economic Analysis
Appendix C: Regulation and Permitting of Algae Farm Siting and Operations
Appendix D: Framework Issues for Use of Wastewater in Algal Cultivation
Appendix E: Regulatory Considerations for Marketing Algal based Food and Feed
Appendix F: Requirements for Marketing a Biofuel
Appendix G: Quality Attributes and Considerations for Trading Algal Oils
ABO 2012 Corporate Members
Information for Stakeholder Comments:
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Additional Reviewers: Amha Belay, Earthrise Nutritionals, Greg Sower, ENVIRON IntI., Tryg Lundquist, California Polytechnic, William Hiscox, WSU
Pullman, ABO Board of Directors representing: Matrix Genetics, BioProcess Algae LLC, Algaedyne Corporation, Accelergy Corporation, MicroBio
Engineering, Inc., The Boeing Company, General Atomics, Scripps Institution of Oceanography, FedEx Express, Los Alamos National Laboratory,
UOP/Honeywell, Fredrikson & Byron Law, Algenol Biofuels, Inc., Sapphire Energy, Wilson Sonsini Goodrich & Rosati, Environmental Biotechnology
Consultants, Ron Pate representing Sandia National Labs and James Collett representing Pacific Northwest National Laboratory.
Executive Summary
Industrial Algae Measurements Version
6.0 is a collaborative effort representing
the collective contributions of over thirty
universities, private companies and national
laboratories over the past five years. This
fully updated October 2013 version offers
detailed recommendations on measurement
methodologies for use across the industry
and roadmaps of the regulatory environments
surrounding different facets of the industry.
This 2013 Industrial Algae Measurements (IAM
6.0) supersedes the 2012 Minimum Descriptive
Language (MDL 5.0) and previous MDLs that
the Technical Standards Committee had
published from 2010 through 2012. Overall,
the industry guidance contained in IAM 6.0
has been slightly broadened in scope with
increased depth and graphical illustration.
Its methodologies continue to equally
encompass heterotrophic, autotrophic, open
pond, photobioreactor and open water
production as well as harvest and conversion
processes for microalgae, macro algae and
cyanobacteria.
In the first section of IAM 6.0 we introduce
ABO’s “Green Box” approach of describing
the industry’s environmental, economic
and carbon footprint via quantifying the
inputs and outputs of an installation. These
input/output measurements systemically
allow for economic projections (through
techno-economic analyses) and sustainability
calculations (through life-cycle assessments).
Green Box inputs include the carbon,
water, energy and nutrients required by the
algae as well as land requirements, process
consumables and manpower requirements
required by the infrastructure. Green Box
outputs include the different classes of algal
products as well as industrial waste emissions
including gas, liquid, and solid discharges.
Together, the measured inputs and outputs
generically carve out the total economic
and environmental footprint of any algal
operation. Identifying this total footprint will
A. Total Infrastructure (Hectare)
B. Total Energy Input (kWh/yr)
C. Total Consumables Input (kg/yr)
D. Total Required Labor (FTEs)
E. Water Input (Liters/yr)
F. Total Nutrient Input (kg/yr)
G.Carbon Input (kg/yr)
become increasingly central in the funding,
regulatory and sustainability review of an
expanding algae industry, and will ultimately
come to define the commercial viability of
specific ventures.
In the IAM 6.0 appendices that follow we
present the methods and language of
algal measurement and provide a guide
to the regulatory environment and other
considerations applicable to the algae industry.
•Appendix A discusses state-of-the-art
methodologies for measuring algal
production of valuable products
at the cellular level.
•Appendix B gives a rudimentary
understanding of Life Cycle Analysis
applicable to the algae industry and
increasingly important in the funding
and government support of programs.
•Appendix C overviews regulatory and
permitting processes applicable to algae
farming and product generation
operations.
•Appendix D discusses the considerations
of using wastewater as a nutrient and
water source for an algae farm.
•Appendix E outlines regulatory process
steps required to consider algae as a
food product, nutraceutical,
colorant or animal feed source.
•Appendix F describes regulatory and
standards considerations needed to
produce a legally marketable
biofuel from algae.
•Appendix G discusses trading rules and
standards applicable to algal oils as they
begin to flow into the commodity
markets now dominated by vegetable
and animal rendered oils.
‘Green Box’ Provides a
Technical, Economic,
& Environmental
Boundary
Accounting for an
Algae Operation’s
Total Yearly Inputs
& Outputs
The Challenge
the Industry Faces
Algal operations will vary in size from
individual bioreactor arrays producing
specialty chemicals and nutraceuticals
to expansive farm scale production of
food products and biofuels.
Accurate
assessment of their future economic and
environmental footprint will be critical to
financing development and performing
environmental life cycle analysis. There is
no harmonized descriptive language set nor
have measurement methodologies been
specifically developed to describe the diverse
technologies being proposed for scaled
algal farms. The lack of a suitable common
language and methodology has created
confusion in expressing attributes and
represents a barrier to industry expansion
until one is adopted.
ABO’s Green Box Solution
The ABO Technical Standards Committee
proposes a set of descriptive language and
measurement methodologies tailored to
the growing needs of our industry across
its diverse technologies, operation size and
product types. ABO’s Green Box approach
manages complexity by measuring and
characterizing process inputs and outputs
only at the point that they would pass through
the boundary wall of an analytical “Green
Box”. This boundary might encompass just an
algal farm or fermentation facility, or it could
further include the plants infrastructure, its
water source or a portion of a biorefinery or
power plant connected to the farm. In this
way, the Green Box descriptive method can
be adapted to compare algal operations
having wholly different inner workings yet
having similar inputs and outputs. The
essential set of input and output variables
required to characterize the economic and
environmental footprint are shown in the
graphic below and are further described
through the balance of this document.
H. Algal Constituent Products (kg/yr)
e.g. Dry algal biomass, protein, oil etc
I. Indirect Algal Products (kg/yr)
e.g. Ethanol, Isobutyraldehyde, fish etc
J. Un-captured Gas Emissions (kg/yr)
e.g. CO
, NO , 2H O, Hydrocarbons etc
2
A note on “algae” versus “algal”:
ABO has adopted the common
parlance of using “algae” when
describing the industry as the “algae
industry” and the ABO organization
as the “Algae Biomass Organization”.
However, the correct scientific usage
applied elsewhere in this document
and recommended to users for
technical and scientific discussions
is as follows: algae is the plural noun
referring to a multitude of cells, alga
is a single cell and algal is the proper
adjectival form.
Support of Life Cycle Analysis: Adoption
of the descriptive language, measurement
and analysis methodologies of IAM 6.0 will
facilitate the uniform conduct of Life Cycle
Analysis (LCA) and techno-economic analysis
within the industry. Additional guidance on
LCA studies may be found in ISO 1404x series
of documents that describe goals, scoping,
quality, transparency and requirements for
data collection, along with guidelines for
the appropriate analysis and presentation
of data. The EPA Renewable Fuel Standards
(RFS2) guidance also provides a template on
how to conduct LCAs for biofuels to meet
various renewable definitions.
Adoption into Peer-Reviewed Research:
The Committee welcomes the voluntary
adoption of IAM 6.0 language and
measurement methodologies into peer
reviewed research.
And likewise the
Committee depends upon peer reviewed
research and its own peer review processes
to form its recommendations. We welcome
growth in academic and industry contribution
to, and participation on our Committee.
Process for Incorporating Stakeholder
Comment:
This IAM 6.0 Guidance
Document is designed to meet the
evolving needs of the algal industry and its
stakeholders. Accordingly, the ABO Technical
Standards Committee invites formal
stakeholder comment on furthering the
scope and specifics of this document. Please
contact ABO directly to log comments and
contributions into the Technical Standards
Industrial Measurement of
Large Scale Operations
The ABO Technical Standards Committee
recommends that when large scale algal
operations are proposed or analyzed, the
following sets of descriptive metrics and
Green Box methodologies are adopted to
uniformly characterize these operations.
By harmonizing upon a common set of
descriptive metrics, the algal industry
will accelerate its growth by eliminating
confusion in the business and life cycle
analysis arena of our industry. By identifying
the sources and characteristics of inputs,
and the intended fates and characteristics
of outputs, we will make the necessary data
available that will allow for upstream and
downstream life cycle, environmental and
techno-economic analysis.
The following “A” through “L” descriptions
of the seven inputs and five outputs of the
Green Box are discussed in accordance
with the diagram above. By knowing
the quantity of inputs and outputs we
can do the basic math. By knowing the
character of the inputs and outputs we
add an understanding of the value flow.
By knowing the upstream source and
downstream fate of inputs and outputs
we further add an understanding of the
sustainability and enduring footprint
of an operation. As the size of an algal
facility increases, the importance of a
comprehensive understanding of the
inputs and outputs expands.
A. Input: Total Infrastructure Area –
Significant amounts of land are typically
needed to cultivate algae, process it into
products and convey input and output
materials (solids, liquids, gasses), power,
energy, vehicles, equipment, and other
machinery around the algal farm and
processing facilities. Total infrastructure
area should encompass every operational
element being analyzed with the Green
Box methodology. Additional components
may include operationally associated, but
off-site, industrial elements, crop fields,
wind farms, solar farms, water treatment
facilities, etc.
Land
Ponds & PBRs
Roads & Pipes
Evaporation Ponds
Buildings
Measurement: Data should include
total footprint area in acres or hectares of
infrastructure includes ponds, PBRs and
fermenters, as well as evaporation ponds,
access roads, pipes and other utilities,
buildings and appropriate portions of
offsite processing or generation facilities
that may appropriately be characterized
as part of the infrastructure of the
overall Green Box analysis. For outdoor
photosynthetic cultivation, the total direct
solar areal capture area of ponds or PBRs
should be broken out if photosynthetic
productivity data is desired.
Source Description: Infrastructure land
should be described as under private
ownership, leased, government, tribal,
reclaimed, conservation reserve program,
or other land use or designation category.
Detail Characteristics: Latitude, altitude,
climate zone, precipitation, slope, soil type
and soil carbon content, indigenous plants
and animals, etc.
B. Input: Total Energy In – Algal
operations may use electricity, fossil fuels,
other chemical energy, steam, etc. that
are imported into the Green Box. In the
case where there is embodied energy in
organic nutrients for heterotrophic algal
production, the nutrient or chemical
energy source should be fully characterized
in the information from the Carbon Input
or Nutrient Input sections to permit future
calculations of upstream environmental
footprint.
Energy
Circulation Pumping
Heating & Cooling
Harvest & Extract
Operations Lighting
K. Liquid Waste Output (Liters/yr)
e.g. Saline or biologic discharge etc
L. Solid Waste Output (kg/yr)
e.g. Biologics, salts, airborne dust etc
Source
Character
Quantity
of input
of input
of input
Green Box’s in-out
descriptive hierarchy
Quantity
Character
Fate of
of output
of output
output
p AIM 6.0 describes an algal operation by surrounding selected operational components with a “Green Box” and then describing the collective whole via the nature of its collective
inputs and outputs.
Measurement: KW-hours (kWh), BTUs, or
other quantity depending of the type of
imported energy should be used. These
can be related to Joules for determination
of overall energy balance. Note that if the
algal operation is a net electricity or other
energy type producer (beyond that energy
embodied in the direct and indirect algal
products) then the Total Energy Input can
be adjusted or made negative to reflect this
net non-algal energy export.
Source Description: Electricity sourced
from fossil coal/gas, biopower or biomass
co-generation, biogas, wind and solar
generation or direct use of fossil resources
as in gasification, etc. Transportation
for energy input includes reference to
transmission lines, pipelines, truck or rail
and distance traveled.
Detail Characteristics: State input
voltage level, phase, and frequency for
electricity as well as other characteristics
that may needed for both fossil and
renewable energy footprint calculation.
C. Input: Total Consumables – The
parts or facilities that must be replaced in
one year of operation. All subcategories
that amount to 10% or more of the total
monetary value of the consumables or
their input mass should be broken out
individually.
Consumables
Replacement Parts
Antibiotics
Reagents
Tools & Vehicles
Measurement: Include dollar value,
description and weight in Kg or tons of
each generalized industrial component in
a form allowing upstream lifecycle impact
assessment.
Source Description: General
identification of source, transportation
distance and information is needed to
calculate upstream footprint in future
analysis.
Detail Characteristics: Cite ability to be
recycled, toxic disposal load, imported vs.
domestic sourcing.
D. Input: Total Required Labor – Algal
operations will need laborers, technicians,
engineers and other special classes of
labor.
Labor
Construction
Maintenance
Operations
Design
Measurement: Provide full time
equivalents of each labor classification
type, even if labor has seasonal variability
so as to allow for economic impact
assessments.
Source Description: Cite if workers are
regionally housed commuters, on-site
housing, locally recruited vs. imported
labor, training site locations, and worker
demographics.
Detail Characteristics: Describe labor
type, education type and source of
required specialized education.
Measurement: Kilograms or tons of
each individual nutrient and its chemical
delivery form should be reported; for
example, NH4NO3 for nitrogen or H3PO4 for
phosphorus.
Source Description: Factory, mine,
wastewater, agricultural waste or other
recycled sources shold be noted and
specify the nutrient transportation distance
for LCA accounting.
Detail Characteristics: Provide fossil
fuel-based manufacturing details, mined,
renewable source, etc.
E. Input: Water In – Water in fresh, saline
and wastewater forms is a basic component
of algal growth media. See Appendix D for
special wastewater considerations. Fresh
water is typically lost through surface
evaporation and may be gained from
precipitation in open systems and may
be lost in closed systems via their cooling
apparatus. The concentrations of dissolved
salts and other chemicals in cultivation
media will increase with evaporation and
recycling.
G. Input: Carbon – CO2, regardless
of source, is typically dissolved in the
growth media of photosynthetic algae to
accelerate growth. However, the carbon
could come from chemical source such as
bicarbonate and others. Heterotrophic and
mixotrophic algae may obtain carbon from
sugars or other organic compounds.
Water
Feed Algae
Evaporation
Process
Drinking
Measurement: Input water volume is
measured in liters or gallons brought into
the Green Box per year. In operations
employing “once-through” cooling
systems, the downstream consumptive and
environmental effects must be taken into
account.
Source Description: Well, pipeline,
river, ocean, lake, surface or groundwater
agricultural allotment, wastewater type,
precipitation, etc.
Detail Characteristics: Total dissolved
solids, mineral type, total organic carbon,
biological oxygen demand (BOD), major
cations and anions, trace metals, bio or
industrial contaminants.
F. Input: Total Nutrients – Algae, like
plants, require macronutrients like
nitrogen, phosphorous and potassium as
well as a host of micronutrients specific to
the species. Carbon has its own separate
category in this Descriptive Language.
Nutrients
Nitrogen
Phosphorus
Micro-nutrients
Sugars
Carbon
Feed Algae
Power Farm
Infrastructure
Consumables
Measurement: Quantify in Kg or tons
per year the quantity of carbon or CO2
imported across the imaginary Green Box
boundary regardless of source and mode
of delivery.
Source Description: Prospectively, pure
CO2 from a pipeline or industrial storage
system, dilute CO2 in flue gas, CO2 drawn
directly from the atmosphere, chemically
bound inorganic carbon in the form of
bicarbonate, or sugars and other simple
organic compounds, either pure or in
mixtures, derived from lignocellulosic
biomass.
Detail Characteristics: Description of
other compounds mixed with the carbon
source, including possible heavy metals
and other contaminants, micronutrients
associated with flue gas (e.g., from coalfired emitter sources), and input pressure
levels if the carbon delivery is in gas or in a
liquid form.
H. Output: Algal Constituent Products*
– Extractable oil, total lipids, protein,
carbohydrate, whole algae, diatomaceous
material, chitin, or any direct constituent of
the organism that is a product.
Algae Oil
Protein
Carbohydrates
Special Materials
Constituents
Measurement: Provide productivity in
kilograms or tons at a specified level of
dryness; the composition of some products
is expected to include ash unless specified
ash-free.
Fate or Destination: Specific use in
fuel, food, durable goods, nutraceutical,
agriculture, industrial chemical, etc.
Detail Characteristics: Detail specific
algal type, harvesting method, level of
biological and chemical purity.
*See Appendix A: Measuring/estimating
algal dry weight and constituents like oil
using small samples.
I. Output: Indirect Algal Products
– Ethanol, extracellular oils, oxygen,
hydrogen, hydrocarbons, remediation
services, shrimp, fish, etc. produced by
organism and sold as a product or benefit.
Ethanol
Fish, Shrimp
Extracellular Oils
Wastewater Clean
Indirect Outputs
Measurement: Kilograms, tons, gallons
or liters of products at specified dryness,
quality or purity level that is exported from
Green Box.
Fate or Destination: Refining into fuel,
human food or nutraceuticals, animal feed
compounding, durable goods agricultural
applications, industrial chemicals
produced.
Detail Characteristics: Species if
appropriate, level of biological and
chemical purity, applicable standards or
specifications are to be documented.
J. Output: Un-captured Gas Emissions**
– CO2, NOx, CO, H2S, H2, O2, water vapor,
volatile hydrocarbons, etc. that are emitted
from any part of the infrastructure. Solid
materials and biologic airborne emissions are
accounted for under solid waste.
Carbon Dioxide
Nitrous Oxide
Water Vapor
Volatile Chems
Un-captured Gas
Measurement: Report PPM or PPB
for regulatory or permitted limits and
kilograms or tons of individual gas
components emitted from whole operation
on annual basis.
Fate or Destination: After emission
to ambient air, disclose the dispersion,
deposition or chemical conversion that
takes place with respect to environmental/
atmospheric conditions.
Detail Characteristics: For example:
Describe whether gas emissions expelled
are components from a Green Box Input
gas stream or are newly created by
processes within the Green Box.
K. Output: Liquid Waste Output***
– Water or other liquids containing levels
of one or more pollutants: Saline/salts,
nutrient, biologics, toxics, heat, suspended
solids, sediment, microorganisms, BOD,
TDS, trace metals or other contaminantcontaining liquid. May also include spent
solvents from extraction processes.
Blowdown Water
Biological Spills
Nutrient Spills
Spent Solvents
Liquid Waste
L. Output: Solid Waste Output****
– Retired parts from Total Consumables
input, sludge, viable biologics, airborne
dust of organic or non-organic
composition, etc.
Retired Parts
Airborne Dusts
Spent Sludge
Pipes & Liners
Solid Waste
Measurement: Note kilograms or tons
whether stockpiled on site, buried onsite or moved offsite. In the case of
viable biologics, note species, genetic
modifications, the dispersion mechanism
i.e. liquid or solid discharge, part of
shipped product, airborne, etc., dispersion
density, human/plant/animal health risks,
and viability/risk of organism spreading
to natural systems or other commercial
aquaculture or agriculture systems residing
outside the Green Box.
Fate or Destination: Use of landfill,
burning, burial, chemical neutralization,
storage, airborne or water dispersal and
downstream effects.
Detail Characteristics: Provide chemical
and biological composition, off gassing,
toxicity, seasonal variations.
**See Appendix B: Total Lifecycle Gaseous,
Liquid, and Solid Waste Outputs
***See Appendix C: Government
Environmental Programs for Regulation
and Control of un-Captured Gas, Liquid,
and Solid Outputs from Algae Operations
****See Appendix D: Framework Issues for
Use of Wastewater in Algae Cultivation
Measurement: Provide PPM or PPB for
regulatory or permitted limits as well
as liquid liters or gallons discharged or
otherwise conveyed from algal production
facility.
Fate or Destination: For water, point
source and non-point source discharge to
surface water, ground water, reinjection,
water treatment, or conveyed/transported
for recovery of useful constituents or
disposal. For liquids such as organic
solvents, appropriate industrial disposal,
storage, or treatment, e.g. purification and
recycling outside the Green Box.
Detail Characteristics: Composition
and concentration of chemical, biological
and other constituents, toxicity, and algal
production process variations should be
reported.
Appendix
Appendix A: Measuring Dry Weight and
Performing Compositional Analysis of Algal Biomass
Uniform methodologies are required to assess the large variety of metrics within the growing algal industry. One of the most pressing and
fundamental areas of standardization is in the measurement of algae productivity and compositional analysis. How can we measure the
quantity of algae in a sample and the type and quantity of constituents that give it market value? These analytical determinations underpin the
measurement of growth rates and value creation, yet uniform measurement of these fundamental attributes has been elusive and historically
poorly described in the literature. Appendix A discusses these challenges and recommends best measurement practices based upon its review
of existing methods while soliciting improvements to these methods from the community on an ongoing basis.
20
50
25
Protein_B
Protein
(% DW)
Reference
15
Reference
Reference
Lab.1
Lab.2
Lab.3
5
5
30
10
40
10
15
LipidsLipids_S
(% DW)
60
Carbs
Carbohydrates
(% DW)
30
20
35
70
Figure 1 clearly illustrates the need for harmonizing methodologies within algal constituent analysis.
Lab.1
Lab.2
Lab.3
Lab.1
Lab.2
Lab.3
p FIG 1: In 2011 three prominent laboratories (Lab.1, Lab.2 and Lab.3) took an identical dried algal sample and analyzed this using prevailing and historical laboratory methods
(gravimetric lipids, colorimetric Lowry protein assay and carbohydrate measurements using phenol sulfuric acid), one of the laboratories carried out the more advanced respective
reference methods (amino acids, monosaccharides by liquid chromatography and direct fatty acid content of whole biomass as measurement for protein, carbohydrates and lipid
content determination respectively). More information and procedure details of this round robin experiment can be found in reference.1 The results showed surprising measurement
variability among laboratories, inaccuracies in prevailing methods relative to reference procedures and have led to a desire to unify analysis methodologies.
The composition of biomass forms a crucial
point in an algal biofuels production process
or operation, and, as demonstrated by
this technical standards document, there
is a conscious effort ongoing to suggest
standard analytical methods so researchers
and industry members in the field are able
to compare processes and track individual
components. The experience of researchers
in the ligno-cellulosic biofuels industry could
be used as a basis for generating similar
standards in algal biomass characterization.
For an algal biofuels process, it is important
to be able to determine the biomass
productivity and algal constituent products
and complete the material balance of
all components during every step of the
operation or process. The main constituents
that make up algal biomass are lipids,
proteins, carbohydrates and ash (including
diatomaceous material). We provide here
suggestions on standardization of the
protocols for quantification of lipids, proteins
and carbohydrates.
1. Dry weight measurements, comparison
of methods for determining productivity
At the basis of any production process
lays productivity and composition
measurements, which are dependent on
both robust dry weight and cell number
calculations. One also should keep in mind
that methods for determining algal dry
weight may be affected by the presence
of significant quantities of contaminating
microorganisms in the growth medium.
Additionally, note that one is typically trying
to assess the equivalent dry weight of algae
in a small sample of liquid medium in order
to indirectly assess the weight of biomass in
a large volume of the same medium. For this
reason, accurate measurement of sample
volume and representative sampling is
essential.
Dry weight can be assessed by “primary
measurements” in which the desired
component is separated and directly
weighed or by “indirect measurements”
such as cell counting or fluorescence, where
the measured quantity is calibrated to
actual mass and used as an analog. Indirect
measurements can be extremely useful
L.M.L. Laurens, T.A. Dempster, H.D.T. Jones, E.J. Wolfrum, S. Van
Wychen, J.S.P. McAllister, et al., Algal biomass constituent analysis:
method uncertainties and investigation of the underlying measuring chemistries., Analytical Chemistry. 84 (2012) 1879–87.
1
because of their typical speed and sensitivity
when calibrated and so both are discussed.
The Technical Standards Committee aims
to provide in this document relatively easy
and inexpensive ways to perform primary
measurement methods that work across
a variety of algae and aquatic biomass.
Subsets of methods are described here
with their respective advantages and
disadvantages:
Filtration: Filter a specific volume of growth
media through a pre-weighed Whatman
GF/F style ultrafine binder free glass fiber
filter. Dry and re-weigh filter on a precision
balance. Repeat drying and weighing until
equal dry weights are obtained.
Advantages: Primary direct measurement
and an easy and inexpensive test of total
non-volatile mass.
Disadvantages: High precision balance
necessary. Vacuum or pressure filtration
equipment needs to be operated at low
pressure. Filtration can easily disrupt
very soft-bodied cells causing significant
loss of material through the filter media.
Unless sample and filter are rinsed with
water before drying, salts from the media
may become included as part of the
measurement. A core assumption of a
drying based methodology is that volatile
components may be lost and unaccounted
for after drying.
Algae change pigment density in response
to specific physiological cues. Presence of
contaminating organisms or suspended
solids can influence turbidity results.
Presence of extracellular components could
significantly alter results.
Cell Count: Count cells with
hemocytometer, Coulter counter, or flow
cytometer (e.g., Fluorescently Activated
Cell Sorter). Note the need to use a primary
measurement method to calibrate cell count
to weight for each algal species.
Advantages: Accurate when calibration
method is rigorous. Very small sample
sizes are acceptable. Hemocytometers and
microscopes are relatively inexpensive.
Disadvantages: Represents an indirect
measurement. This approach can be time
intensive if done manually. The use of a
Coulter Counter or flow cytometer is easier
but these instruments are expensive and rely
upon calibration with a direct measurement
method. Cell counting, especially with a
Coulter counter or flow cytometer, may
be limited by cell size and complicated by
large cell size distribution or by filamentous,
multicellular or colonial algal morphology.
Organic Carbon Content: A variety of C
analyzers are available to measure total
organic carbon in aqueous samples. Solid
phase CHN analyzers will measure total
carbon on a dried GF/F filter or a dried pellet.
There is need to use primary method to
calibrate to cell weight for each algal species.
Advantages: Accurate even for small sample
size if calibration is accurate.
Disadvantages: Indirect Measurement,
expensive equipment and C/N ratio in algal
cells changes with time of day and growth
conditions.
2. Compositional Analysis of Algal
Biomass, Lipids, Carbohydrates and
Protein
Characterization of algal biomass consists of
the accurate measurement of lipids, proteins
and carbohydrates as the major constituents
of all biomass samples. The degree to
which these are characterized primarily
depends on the information required and
different methods provide different types of
information.
2.1 Algal lipids vs. extractable oils
vs. fuel fraction
Traditionally, lipids have been measured
as a gravimetric solvent extraction yield.
However, different procedures and types of
solvents have resulted in inconsistent lipid
Centrifugation: Spin a volume of culture
in a pre-weighed centrifuge tube. Remove
supernatant liquid. Dry and weigh pellet
plus tube.
Advantages: Primary measurement and
easier measurement of heavier-than-media
cells and particles.
Disadvantages: Centrifuge and significantly
high-resolution balance can be expensive
and small pellet size can severely
compromise accuracy of the results. Core
assumption that all components to be
measured are heavier than media may
influence result if light-weight components,
such as lipids from lysed cells, are poured off.
Turbidity: Can be measured with several
instrument types (e.g., spectrophotometer,
turbidimeter) at selected wavelengths.
Note that one must compare turbidity
measurements at several cell densities
with a direct mass measurement method
to determine the turbidity to weight
relationship for each algal species.
Advantages: Good for small volume samples.
Fast measurement and low cost sensor
technology make this method useful for
continuous monitoring of growth dynamics.
Disadvantages: Indirect Measurement.
yields.2, 3 The completeness of extraction
and composition depends on the biology
of the alga and the physiological conditions
experienced by the organism, as well as
the compatibility of the solvent polarity
with the lipid molecule polarity and
extraction conditions used. Inevitably, the
extractable oil fraction will contain non-fuel
components (e.g. chlorophyll, pigments,
proteins, and soluble carbohydrates). Thus it
may be necessary to assess the fuel fraction
of these isolated oils (i.e. fatty acid content
of extracted lipids) by transesterification
followed by quantification of the fatty
acid methyl esters (FAMEs). Due to the
large number of variables, it is difficult
to standardize an extraction-based lipid
quantification procedure. Furthermore,
any extraction-based lipid quantification
procedure is not necessarily related to a
production-relevant extraction process and
may not provide industrially applicable
information. However, if the relative
composition of intact lipids is required,
e.g. polar versus neutral lipid content, an
extraction process may be the only way to
isolate lipids from the rest of the biomass.
There are two typical extraction systems
currently in use across algal biomass
analytical laboratories; either conventional
Soxhlet extractors or the more recently
developed pressurized fluid extraction
systems (as in the commercially available
Accelerated Solvent Extractor, Thermo
Scientific). The solvent systems used for
extraction will influence the gravimetric
yield and composition of the lipid
fraction,2,4,5 but this is the only manner in
which total lipid composition data can be
obtained. After extraction, total lipids can be
further analyzed by liquid chromatography
to quantify for example the TAG content.6
As an alternative to extraction, there is
a growing emphasis on quantification
of lipids through a direct (or in situ)
transesterification of whole algal biomass.
The process consists of either a two-step
basic followed by acid hydrolysis of the
biomass 7,8 or a single step acid catalysis 5,9,10
followed by a transmethylation of the fatty
acids and quantification of the FAMEs by
gas chromatography (GC). These procedures
have been demonstrated to be robust
across species and their efficacy is less
dependent on the parameters listed above
that influence an extraction process. One
disadvantage is that no information on the
origin of the lipids is available, e.g. relative
level of neutral and polar lipids, for which
additional instrumentation is needed such
as liquid chromatography. Several reports in
the literature and in the AOAC (Association
of Official Analytical Communities)
official methods are suggesting in situ
transesterification as the lipid quantification
procedure of choice for algal biomass.5,7-10
The applications of the respective methods
are varied; reference9 was established as
a sub-microscale procedure for very small
quantities (< 1mg) of algae and allows for
single cell calculations by extrapolation to
culture volumes. The methods detailed in
references 5,7,10 were developed for freeze
dried biomass (>10 mg) while still allowing
for relatively high-throughput analyses (100500 samples per week). The AOAC official
method 8 was established as a method for
M.J. Griffiths, R.P. van Hille, S.T.L. Harrison, Selection of Direct
Transesterification as the Preferred Method for Assay of Fatty Acid
Content of Microalgae, Lipids. 45 (2010) 1053–1060.
the fatty acid determination in encapsulated
fish oils, but has been used extensively on
algae and forms the basis of the work shown
in reference.7 A study of increasing biomass
quantities on the precision of the total
FAME content measured using a modified
reference7 method indicated that higher
precision could be achieved by increasing
the biomass, presumably by eliminating
weighing inaccuracies. The relative standard
deviation of replicate measurements
dropped significantly at biomass quantities
of over 10 mg11 and Gardner et al.,
unpublished data. When limited by very
small sample sizes, an alternative high
precision approach is to use cell number
rather than biomass for assessing fatty acid
concentration.9
2.2 High-throughput in vivo
measurement of lipid content using
lipophilic dyes
In addition to the procedures listed
previously, there has been an emphasis
to accelerate the quantification of lipids.
Researchers want to tailor the analyses to
the screening of thousands of individual
strains in bioprospecting or metabolic
engineering projects. These highthroughput methodologies are based on
hydrophobic (lipophilic), fluorescent dyes,
such as Nile Red10,12 and BODIPY.13,14 As
7
AOAC, Analysis of Fatty Acids, in: Official Method 991.39,
1995.
8
N.W. Bigelow, W.R. Hardin, J.P. Barker, S.A. Ryken, A.C. MacRae, R.A.
Cattolico, A Comprehensive GC-MS Sub-Microscale Assay for Fatty
Acids and its Applications, Journal of the American Oil Chemists
Society. 88 (2011) 1329–1338.
9
E.J. Lohman, R.D. Gardner, L. Halverson, R.E. Macur, B.M. Peyton,
R. Gerlach, An efficient and scalable extraction and quantification method for algal derived biofuel., Journal of Microbiological
Methods. 94 (2013) 235–244.
10
R.D. Gardner, K.E. Cooksey, F. Mus, R. Macur, K. Moll, E. Eustance,
et al., Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom
Phaeodactylum tricornutum, Journal of Applied Phycology. 24
(2012) 1311–1320.
11
K.E. Cooksey Guckert, J.B., Williams, S.A., Callis, P.R., Fluorometric determination of the neutral lipid content of microalgal
cells using Nile Red, Journal of Microbiological Methods. 6 (1987)
333–345.
12
T. Govender, L. Ramanna, I. Rawat, F. Bux, BODIPY staining, an
alternative to the Nile Red fluorescence method for the evaluation
of intracellular lipids in microalgae., Bioresource Technology. 114
(2012) 507–11.
13
M.S. Cooper, W.R. Hardin, T.W. Petersen, R.A. Cattolico, Visualizing
“green oil” in live algal cells., Journal of Bioscience and Bioengineering. 109 (2010) 198–201.
14
J.B. Guckert, K.E. Cooksey, L.L. Jackson, Lipid solvent systems are
not equivalent for analysis of lipid classes in the mciroeukaryotic
green-alga, Chlorella, Journal of Microbiological Methods. 8 (1988)
139–149.
the dyes are absorbed by the lipids the
sample fluorescence intensity increases
proportionally and this principle has been
used extensively in the screening for higher
lipid producing cells among thousands of
candidates. Note that BODIPY staining may
not be a substitute for Nile Red in semiquantitative fluorescence measurements
of total lipids as the dye does not exhibit
a Stokes wavelength shift when binding
to hydrophobic areas of an algal cell, such
as neutral lipid. Furthermore, although
fluorescent dyes are a powerful and
potentially high-throughput approach for
screening lipid-producing cells, caution
has to be taken with the interpretation of
fluorescence results from both BODIPY and
Nile Red dyes as quantitative indicators due
to the possibility of inconsistent dye-uptake
between different algal cells.
In addition, high throughput or high speed
methods may be required to track changes
in lipid content that occur across light-dark
cycles. During light cycles lipids accumulate.
During cell division and dark cycles these
lipids are consumed by the cells for their
own metabolic needs.
2.3 High-throughput measurement of
algal biomass composition using infrared
spectroscopy
Infrared (IR) spectroscopy coupled with
chemometrics to identify high lipid
producing strains from an algal population
is a quantitative compositional analysis
method that can contribute to quantitative
screening of algal biomass.15,16 One
advantage of IR technology over the
hydrophobic, fluorescent dyes such as
Nile Red and BODIPY is that the IR-based
quantification is not dependent on the
permeability of the cell walls to the dyes that
has been reported to affect the fluorescence
signal from lipids.
2
C. Bigogno, I. Khozin-Goldberg, S. Boussiba, A. Vonshak, Z. Cohen,
Lipid and fatty acid composition of the green oleaginous alga
Parietochloris incisa, the richest plant source of arachidonic acid,
Phytochemistry. 60 (2002) 497–503.
3
S.J. Iverson, S.L.C. Lang, M.H. Cooper, Comparison of the Bligh and
Dyer and Folch methods for total lipid determination in a broad
range of marine tissue, Lipids. 36 (2001) 1283–1287.
4
L. Laurens, M. Quinn, S. Van Wychen, D. Templeton, E.J. Wolfrum,
Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification, Analytical and Bioanalytical Chemistry. 403 (2012) 167–178.
5
K.M. MacDougall, J. McNichol, P.J. McGinn, S.J.B. O’Leary, J.E.
Melanson, Triacylglycerol profiling of microalgae strains for
biofuel feedstock by liquid chromatography-high-resolution mass
spectrometry., Analytical and Bioanalytical Chemistry. 401 (2011)
2609–16.
6
p FIG 3: Lipid bodies fluoresce green (stained with BODIPY 505/515). Difference in size
and number of lipid bodies among Chromista: A. Emiliania huxleyii, 5μm; B. Ochromonas
danica, 8μm; C. Phaeodactylum tricornutum, 20μm; and D. Aureococcus anophagefferen,
4μm . (Dong, Hardin and Cattolico, unpublished data).
FIG 4: TAG lipids are fluorescing yellow in Amphora coffeaeformis when stained with
Nile Red. The micrograph shows the results when a culture enters stationary phase. If
centrifugation is used to harvest cells, the extracellular TAG associated with the right
hand cell will not be included in the TAG yield. This will be missed if cultures are not
inspected microscopically. Photo courtesy Keith Cooksey MSU u
Infrared spectroscopy (mid- and near IR)
offers the possibility of a rapid and sensitive
technique for determining the composition
of algae because organic compounds’
infrared vibrations directly follow Beer’s
law and can be used for quantitative
determinations. Mid- and near-IR are able
E. Wolfrum, L. Laurens, Rapid compositional analysis of microalgae by NIR spectroscopy, NIR News. 23 (2012) 9.
15
L.M.L. Laurens, E.J. Wolfrum, Feasibility of Spectroscopic Characterization of Algal Lipids: Chemometric Correlation of NIR and
FTIR Spectra with Exogenous Lipids in Algal Biomass, BioEnergy
Research. 4 (2010) 22–35.
16
collected, multivariate statistical software
packages are used to compare the new
spectra against a calibration spectra set
and quantitative regression models are
used to return the compositional data of
the unknown samples. The quality of the
predictions is highly dependent on the
quality of the original calibration models and
significant value is placed on establishing
robust models based on accurate and
precise wet-chemical data using state-of-the
art procedures listed above.
2.4 High-throughput in vivo measurement
of neutral lipids in algae using 1H-NMR
A rapid, non-destructive method for in vivo
analysis of oil content in live algal cultures
by 1H Nuclear Magnetic Resonance (1H NMR)
has recently been developed.17 The method
is specific for neutral lipids (“TAGs”, including
free fatty acids and mono-, di- and tri-acyl
glycerides) stored in cell lipid bodies. Less
than 1ml of algal culture is required for
analysis, and the measurement takes only
minutes on commonly available (300MHz
or greater) NMR spectrometers. Virtually
no sample preparation is required, and
drying is unnecessary. However, the culture
medium can be isolated from algal cells
P.T. Davey, W.C. Hiscox, B.F. Lucker, J. V. O’Fallon, S. Chen, G.L.
Helms, Rapid triacylglyceride detection and quantification in live
micro-algal cultures via liquid state 1H NMR, Algal Research. 1
(2012) 166–175.
17
quantitatively to determine the amount of
lipids to algal biomass from different species.
By combining the measured lipid content
with the spectra using multivariate statistical
approaches, predictive calibration models
can be built. NIR spectra were correlated
with the increasing concentration of lipids,
and could distinguish between neutral and
polar lipids. Recently a similar approach
was taken, where near-IR spectra of a set
of biomass samples were used to build
quantitative prediction models of the full
biochemical composition of three microalgal
strains.
This approach is a powerful and quantitative
approach that can take the full quantitative
biochemical analysis of algal biomass from
several days down to a minute on a fraction
of the material needed for traditional
chemical analyses. The only requirement
for quantitative prediction of a new set of
materials is a robust predictive model of
NIR spectra based on a fully characterized
calibration set of samples.
In a typical analysis, spectra are taken
from freeze dried algal biomass, added
to each well of a 96 well plate for near-IR
spectroscopy or placed on the diamond-ATR
cell of a mid-IR system. After the spectra are
susceptibility differences between the liquid
oils stored in lipid bodies and the water
matrix of the culture sample by use of High
Resolution Magic Angle Spinning (HRMAS)
1
H NMR (Figure 9.C), which gives results
similar to static liquid state NMR spectra of
isolated lipids in solution (Figure 9.D).
by centrifugation, and analyzed separately
to reveal the presence of any extracellular
organic components in the matrix, and dry
weights may still be obtained, if desired, in
order to assign a percent neutral lipid by dry
weight value. The lower limit of detection
of neutral lipids in a culture by this in vivo
method is about 30 µg/ml.
In a typical analysis, a <1ml sample of algal
culture is placed in an NMR tube (Figure
8), and a coaxial capillary insert containing
a calibrated reference solution is inserted
into the tube. The assembly is then placed
in the magnet of a NMR spectrometer
and a spectrum is taken. Since 1H NMR is
an inherently and directly quantitative
measurement of all the observable protons
in the sample, the NMR signals between
0.25 and 2.85 ppm due to TAGs (Figure
9.B, Figure 10, Static proton NMR) can be
integrated and compared to the reference
signal (calibrated standard TMSP in D2O) at
0.0ppm. To calculate the concentration of
neutral lipid in the aqueous culture sample,
a representative “model TAG” is derived,
based on fatty acid constituent analysis of
isolated lipid from the same alga. The model
TAG provides an average molecular weight
and proton count, which is used to convert
the neutral lipid/reference standard integral
ratio to mass or concentration units. Results
are generally reported in the form of weight
of neutral lipid per volume (w/v) of culture
Total NMR %
Error
Time (hrs)
0
24
48
72
96
120
144
168
192
216
Sensitivity of the measurement increases
with increasing magnetic field. However,
high field NMR instrumentation (500MHz
spectrometers and above) may only be
available at relatively large institutions, such
as major universities, due to their high cost
(>$500K). Fortunately, equivalent results
may be obtained on a 300MHz system
in many cases by collecting data over a
longer period of time by signal averaging.
Typically, sufficient signal-to-noise ratios
can be obtained in around 15 minutes on
a well tuned 300MHz system, compared
to 1 minute or less for high oil content
cultures on a 500MHz or greater instrument.
Currently, a refurbished superconducting
300MHz NMR spectrometer can be
purchased for around $65,000 - $70,000,
including installation. Work is in progress
to extend the method to lower field, lower
cost instruments for production/process
monitoring applications.
3.7 +/- 2.1
12.1 +/- 1.6
30.8 +/- 1.8
75.8 +/- 2.2
110.4 +/- 3.9
142.5 +/- 3.9
158.8 +/- 5.2
180.2 +/- 4.8
212.6 +/- 5.1
222.8 +/- 6.0
(making it useful for process monitoring).
However, w/w percentages of dry biomass
can be derived by isolating and drying cells
from a known volume of culture to get a dry
weight. The 1H NMR method is amenable
to continuous sampling of single cultures
(Figure 10) or to high throughput analysis of
multiple cultures.
The in vivo 1H NMR method is specific for
neutral lipids. Membrane lipids are not
measured by this method, in contrast to
57.6
13.5
5.7
2.9
3.5
2.7
3.2
2.6
2.4
2.7
77.6 +/- 3.9
107.1 +/- 5.3
144.1 +/- 7.2
190.5 +/- 9.5
210.3 +/- 10.5
255.2 +/- 12.7
271.1 +/- 13.5
305.0 +/- 15.2
302.9 +/- 15.1
331.8 +/- 16.4
FAME analysis by GC/MS, which measures
total lipid content from all sources within
the cells. The NMR method’s specificity lies
in the relative differences in the molecular
motions of the storage lipids (high) and the
immobile membrane lipids. Signals from
membrane lipids (phospholipids, etc.) are
broadened to the extent that they become
invisible to liquid state NMR. The broad but
observable signals for neutral lipids seen
in “static” NMR measurements (Figure 9.B)
can be sharpened by removing magnetic
Anticipated applications of algal lipid 1H
NMR include prospecting or screening
of oleaginous algal cultures, process
optimization studies, and process
monitoring and control in large culturing
operations. The method is complimentary
to FAME-GC methods, including direct
transesterification, and can be used to
distinguish neutral lipid production
from lipids derived from cell membrane
components. Neutral lipid concentrations by
1
H NMR have been found to be consistently
lower than total lipids by FAME GC (total
biodiesel potential) for the same culture
samples throughout the oil accumulation
stage. However, the difference in the two
values becomes smaller at high neutral lipid
concentrations (see Table 1)
2.5. Consideration of Chrysochromulina
sp. to provide a lipid analysis standard for
the industry
Unlike other well-established food and
oil commodities, presently no universal
reference standard exists for use in the
algal oil industry. Universal adoption of a
reference standard provides commercial
sites and laboratories with a common
platform to compare the fatty acid
composition of different algal strains grown
under various environmental conditions,
and subjected to different oil recovery
for comparing 20 taxonomically diverse
algal cultures that were grown under
identical physiological conditions and
analyzed for fatty acid content using a
micro-GC/MS analytical technique.9 To
compare fatty acid content and profiles
among algal taxa, representatives of 7 algal
genera (Chrysophyceae, Pavlovophyceae,
Pelagophyceae, Pinguiophyceae,
Prymnesiophyceae, Raphidophyceae, and
Xanthophyceae) were examined. Resulting
data is shown in Figure 12. Pie chart
size depicts total fatty acid productivity
(mg/L) while sector size within the chart
is proportional to the amount of a specific
fatty acid present in the sample. The
reference standard Chrysochromulina sp.
generated both the amount (17.1 mg/L)
and fatty acid profile anticipated. This result
verifies the effectiveness of the processing
and analytical (Black Box of Figure 11)
techniques chosen for the analysis and
show that both total productivity and the
amounts of specific oil type produced vary
widely among algal taxa. In summary, use
of a Chrysochromulina sp. standard grown
under standard conditions could provide
a consistent calibration reference for
comparing total oil recovery and oil profiles
in other alga species. (* The taxonomic
nomenclature of this alga is under revision
to Chrysochromulina tobin.)
methods (FIG 11). A laboratory generated
natural matrix standard has two distinct
advantages: (a) as a reproducibly generated
standard, it can supplant conventional
reference products that vary markedly
among production batches (e.g. the fatty
acid composition of conventionally used
Menhaden oil can be affected by the species
of fish selected, seasonal variations in fish
body oil complement, region of fish harvest,
and oil extraction methodology); and (b) it
will help in the identification and elimination
of errors in lipid extraction, derivatization
and analytical techniques.
The newly identified haptophye strain,
Chrysochromulina sp.* (Haptophyceae) has
been developed as a reference standard for
algal fatty acid analysis.18 This laboratory
optimized alga is amenable to this purpose
because: (a) as a soft-bodied organism, it
is readily susceptible to all conventional
disruption and fatty acid extraction
techniques; (b) it has a high fatty acid
content (~40% dry weight); (c) it’s growth
response and lipid profiles are highly
reproducible; (d) unlike many algae that
have limited fatty acid distributions, cells of
this organism contain a broad representation
of both saturated and unsaturated fatty
acids ranging from 14 to 22 carbons long
(C14 to C22) (Figure 12). Chrysochromulina
sp. has been used as a reference standard
2.6 Algal carbohydrate measurements
Carbohydrates can comprise a significant
portion of algal biomass and thus their
accurate quantification is crucial to
determining the feasibility of utilizing
an algal species for specific biofuel
and co-product pathways as well as for
completing a summative mass closure.
Algal biomass carbohydrate determination
is based on an analytical hydrolysis step to
release polymeric carbohydrates to their
monosaccharide constituents (Figure 13).
There are historical methods based on a
rapid phenol sulfuric acid method, which
claim to hydrolyze and react quantitatively
with the carbohydrates in solution.19, 20
However, the phenol-sulfuric acid method
is notoriously variable and not all sugars
exhibit a similar colorimetric response and
thus some carbohydrates could cause an
over- or underestimation if a calibration
is performed based on one neutral
monosaccharide like glucose.1, 19
M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substance,
Analytical Chemistry. 28 (1956) 350–356.
19
N. Bigelow, J. Barker, S. Ryken, J. Patterson, W. Hardin, S. Barlow,
et al., Chrysochromulina sp.: A proposed lipid standard for the algal
biofuel industry and its application to diverse taxa for screening
lipid content, Algal Research. (2013) 1–9.
18
T. Masuko, A. Minami, N. Iwasaki, T. Majima, S.I. Nishimura,
Y.C. Lee, Carbohydrate analysis by a phenol-sulfuric acid
method in microplate format, Analytical Biochemistry. 339 (2005)
69–72.
CH2OH
OH
HO
O
OH
O
OH
HO
O
OH
O
Chemicals
H H
Enzymes
HO OH
n
Alternative carbohydrate quantification
procedures involve sequential hydrolysis
of carbohydrate polymers in algae and
identification and quantification of
the monomers via liquid (HPLC) or gas
H
O
H
H OH
OH
chromatography (GC, as alditol acetates).21
Because of the use of chromatography,
these procedures are likely to be more
accurate and will also provide a relative
monosaccharide composition of the algae.
There are reports in the literature, but a
comprehensive comparison and test of
robustness across strains is currently lacking.
As a subset of carbohydrates, starch
measurement is among the routine
compositional analysis methods and
represents a common storage carbohydrate
content in algae. This method is selective
for alpha 1,4 linked glucan due a specific
enzymatic hydrolysis step, which are known
to be present in some algal strains but not in
others. The method of biomass preparation
and enzyme assay kit highly affects the
measurement.1 A protocol that has been
demonstrated to give accurate and precise
starch determination on a variety of strains is
detailed in reference.22 Alternative methods
exist and can be considered equally valid
after a careful consideration of the accuracy
20
D. Templeton, M. Quinn, S. Van Wychen, D. Hyman, L.M.L. Laurens, Separation and quantification of microalgal carbohydrates,
Journal of Chromatography A. 1270 (2012) 225–234.
21
www.megazyme.com/downloads/en/data/K-TSTA.pdf, Total
starch assay procedure (amyloglucosidase/ alpha-amylase
method), 2011 (2011).
22
efficacy of cell fractionation (solubilization
of cellular proteins). Improvements have
been made by inclusion of NaOH digestion
of algal biomass and proteins, where
the colorimetric protein determination
corresponds better with the measured
amino acid content. However, a detailed
investigation of the colorimetric data against
amino acid and nitrogen conversion data
indicates a species and growth condition
dependent variability that underpins up to
3-fold differences between the colorimetric
and the amino acid data [NREL, unpublished
data]. Some of the variability that has been
observed between spectrophotometric and
alternative methods speaks to the need for
a careful consideration with respect to the
utilization of data from spectrophotometric
assays.
and precision of the method.
2.7 Algal protein measurements
Protein in algal biomass is a third important
constituent and can make up to half of the
dry biomass weight. In the context of algal
biomass as the basis of food or feed, an
accurate determination of protein content
and the biomass amino acid composition
is imperative to the determination of the
biomass quality.
Protein content in algal biomass can be
quantified using two common procedures;
colorimetric23 and a nitrogen ratio
calculation based on measuring elemental
nitrogen and applying an algae-specific
nitrogen-to-protein conversion factor
to measured total nitrogen content in
biomass.24 More recently, a fluorometric
measurement procedure of algal protein is
being developed and offers the advantages
of requiring a minute amount of biomass,
excellent specificity, compatibility with
a wide suite of reagents and a high
throughput potential. The colorimetric and
fluorometric procedures can be susceptible
to interferences from non-protein cellular
components as well as extraction buffer
constituents, and are highly dependent on
the protein standard used for calibrating
the absorbance/fluorescence values. The
measurements are also dependent on
Calculating protein content using a
nitrogen-to-protein conversion factor has
proven to be a robust representation for
whole biomass protein measurement.
Measuring elemental nitrogen is based
on high-temperature combustion and is
much less susceptible to interferences. An
algal biomass-specific conversion factor
was calculated from the typical amino acid
composition of 12 species of algae grown
under different conditions.24 An overall
average ratio factor of 4.78 grams of algal
protein to detected grams of elemental
nitrogen has been used successfully for
algal protein quantification. However,
variation in the non-protein, nitrogenous
compounds between different strains and
growth conditions of algae will affect the
applicability of the averaged conversion
factor and a detailed investigation of a novel
and generally applicable factor is underway
(Figure 14). Amino acid composition of
algae is perhaps the most accurate protein
determination and official AOAC standard
procedures have been published for the
quantification of acid hydrolyzed amino acid
determination.25 A validation of nitrogento-protein conversion should be carried
out based on amino acid data for each new
strain included in commercial or academic
development.
2.8 Constituent Analysis Summary:
As a summary an overview of the
recommended analytical procedures for
the characterization of commonly reported
constituents in algal biomass is shown in
Table 2
3. Measurement of volatiles and semivolatiles in algal cultures
There are several different approaches
that are suitable for determination of
volatile and semi-volatile chemicals
present in algal cultures. These analytes
may be naturally occurring compounds
or products or byproducts present as a
result of strain development activities. Gas
Chromatography with Headspace Sampling
and Flame Ionization Detection (GC-HS-FID)
comprises an effective method for volatile
measurement. It is the preferred method
for rapid and high throughput analysis of
algal culture volatiles since algal samples
can be directly placed in a vial with little to
no preparation. A typical GC-HS-FID system
having an automated Headspace Sampler is
pictured below.
Volatile components from complex sample
O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein
Measurement with Folin Phenol Reagent, Journal of Biological
Chemisty. 193 (1951) 265.
24
G
S
Volatile
analytes
Sample, dillution solvent
& matrix modifer
The typical sample is usually in the form
of a liquid or solid in combination with
a dilution solvent or a matrix modifier.
Once the sample phase is introduced
into the vial and the vial is sealed, volatile
components diffuse into the gas phase
until the headspace has reached a state of
equilibrium, as depicted by the arrows in the
figure above. The gaseous sample is then
removed from the headspace with a syringe
and injected onto the GC column. In all
cases, adequate headspace of at least 50%
of vial volume is required for the gas phase
sample to freely accumulate.
acetaldehyde, methanol, acetonitrile, ethyl
acetate, methyl ethyl ketone and others.
This modified method is valid for both
freshwater and saltwater media with or
without buffers, antibiotics, trace metals
and other non-volatile components. The
measureable range for liquid phase ethanol
extends from 0.0001% (v/v) to 0.1% (v/v)
and for isopropanol and acetone extends
from 0.00005% (v/v) to 0.1% (v/v). These
values are based on a 2 mL volume of algae
having a growth medium OD of about 1.0.
Samples of 10 mL are crimp sealed in glass
vials having an aluminum cap and septum
appropriate for the automated Headspace
Sampler. If smaller or larger volumes are
desired then reference standard liquids of
the same volume should be prepared to
match the volume.
Varying error effects (Table 3) can occur
due to the salting effect that increases
vaporization of volatile solvents in solutions
by reducing solubility of the volatile
component.
Commercially available volatile
measurement check standards for blood
alcohol dissolved in water can be used to
calibrate equipment used for algal volatiles
measurements. Cerilliant (Sigma Aldrich)
BAC standards are an example of traceable
standards readily available for this purpose.
A slightly modified form of the Blood
Alcohol Content (BAC) method26 can be used
to quantify low levels of volatiles present
in solution using gas chromatography with
flame ionization detection. This process
requires heating to volatilize the compounds
from the matrix, and therefore is not
concentration dependent. This method can
detect volatile and semi-volatile molecules
including ethanol, isopropanol, acetone,
23
S.O. Lourenço, E. Barbarino, P.L. Lavín, U.M. Lanfer Marquez, E.
Aidar, Distribution of intracellular nitrogen in marine microalgae:
Calculation of new nitrogen-to-protein conversion factors, European Journal of Phycology. 39 (2004) 17–32.
mixtures are isolated from non-volatile
sample components in the headspace of a
sample vial. Headspace gas chromatography
is most suited to the analysis of small
molecular weight volatiles in samples as
they are efficiently partitioned into the
headspace gas volume from the liquid or
solid matrix sample. Higher boiling point
volatiles and semi-volatiles may not be
detectable with this technique due to their
low partitioning into the gas headspace.
However, in most cases, the addition of
heat and/or salts can lower the Partition
Coefficient (K) by reducing gas solubility.
Partition Coefficient (K) = Cs/Cg where: Cs
is the concentration of analyte in sample
phase and Cg is the concentration of analyte
in gas phase.
R.L. Firor, C.-K. Meng and M. Bergna, Static Headspace Blood
Alcohol Analysis with the G 1888 Network Headspace Sampler.
Agilent Application Note 5989-0959. (http://cp.chem.agilent.com/
Library/applications/5989-0959EN.pdf )
26
25
AOAC, Amino Acids in Feeds, in: Official Method 994.12, 1997.
Appendix B: Basics of Lifecycle and Techno-Economic Analysis
Appendix C: Regulation and Permitting of Algae Farm Siting and Operations
Lifecycle Assessment (LCA) and TechnoEconomic Analysis (TEA) are used to assess
the total environmental, energy and financial
footprint of a manufacturing process. With
respect to algae, the manufacturing process
commonly being analyzed will include the
steps of building an algal farm, powering
and provisioning the mass production of
algae on that farm, converting or using
algae to produce products like food or fuel,
delivering those products to market, and
the use of the product as, for example, a
fuel that is burned with release of CO2 and
other pollutants. Each step has its own LCA
footprint and an overall LCA process will
clearly define what processes are within its
boundary or scope of analysis.
Most countries have an established body
of environmental laws and regulations to
which algal production operations will be
subject. Such programs typically regulate
at some level one or more of the following:
gaseous emission or air pollutants, water
pollution or discharges to water, solid and
hazardous waste handling and disposal,
facility siting and permitting, and handling
of toxic substances. Additionally, some
countries have regulatory requirements that
relate to micro-organisms and algae, with
respect to their production, importation,
genetic modification, or processing for both
R&D and commercial activities, as well as
their release to the environment. State and
local regulatory authorities will also likely
have additional regulations that will need to
be addressed on a nuanced state-by-state
basis.
An LCA process would minimally involve
determining the energy, carbon and water
balance associated with producing a unit
of product such as a gallon of fuel. For
example, the amount of energy embodied in
an algae based fuel compared to the fossil or
alternative energy required to manufacture
it. In addition, it could evaluate total
carbon emissions or use of water resources
compared to other fuels. It could also
factor in the waste products, air emissions,
raw materials and manpower inputs that are
also part of a complete LCA analysis. An LCA
analysis may assist in determining eligibility
for government incentive programs,
evaluating the environmental impact of
farm operations, projecting economic
performance or performing a Resource
Assessment (RA). The latter, RA is used to
calculate the total amount of fuel or other
product able to be manufactured using a
specific process given the amount of input
resources available within a specific area, or
inform the TEA and LCA analyses of the need
to bring resources to the cultivation facility
from remote locations. In all cases, the
importance of uniform approaches to these
analyses is increasing as the algae industry
seeks to rapidly develop, finance, and build
out its operations.
LCA and TEA analysis tends to be
complex, not only because of the many
inputs, outputs and inter-linkages that
are involved, but also because algal
product manufacturing processes vary
widely and continue to be developed.
The standardized GREET LCA analysis
tool developed by Argonne National
Laboratories has been adapted to over 100
feedstock-to-fuel pathways yet continues
to require adaptation to accommodate the
diversity of algal based fuels
“Life Cycle Analysis of AlgaeBased Fuels with the GREET
Model.” 1 Argonne, NREL and
the Pacific Northwest National
Labs convened a workshop
to assist with harmonizing
the analyses of algal oil based
fuels. Their June 2012 report
“Renewable Diesel from Algal
Lipids: An Integrated Baseline for
Cost, Emissions, and Resource
Potential from a Harmonized
Model” 2 provides an excellent
overview of the LCA and TEA for
lipid based biodiesel. However,
fuels from algae can also be
derived from non-lipid pathways
including the collection of ethanol,
hydrogen, isobutyraldehyde, oils and
other chemicals exuded by algae in-situ.
These can be extracted from algal fluids
or photobioreactor headspaces and then
converted into fuel and other high value
industrial commodities. In one example,
Georgia Institute of Technology and Algenol
Biofuels published “Life Cycle Energy and
Greenhouse Gas Emissions for an Ethanol
Production Process Based on BlueGreen Algae” 3 detailing an LCA based on
producing ethanol from blue-green algae in
a closed loop process that does not require
harvesting of algae or extraction of oil.
ABO’s Green Box Approach is designed to
provide a highly flexible framework for
generating input data for LCA/TEA analysis
that anticipates both these existing, and
future, pathways for the algae-based
production of food, fuel and high value
chemicals. By aiming to describe in a
comprehensive fashion the many inputs
and outputs that occur in algal operations
and identifying the methodologies required
to measure these data, the ABO Technical
Standards Committee is supporting the
algal industry and its need to perform
harmonized LCA and TEA assessments as
both evolve.
The IAM 6.0 document discloses that during
algal production and processing operations,
Life Cycle Analysis of Algae-Based Fuels with the GREET
Model http://www.istc.illinois.edu/about/SeminarPresentations/20111102.pdf
1
Renewable Diesel from Algal Lipids: An Integrated Baseline for
Cost, Emissions, and Resource Potential from a Harmonized Model
http://www.nrel.gov/docs/fy12osti/55431.pdf
2
Life Cycle Energy and Greenhouse Gas Emissions for an Ethanol
Production Process Based on Blue-Green Algae http://pubs.acs.org/
doi/pdf/10.1021/es1007577
3
gaseous, liquid, and solid emissions can
include indirect and direct greenhouse
gases emissions (CO2 , NOx, water vapor,
etc.) associated with the production of
input energy and materials as well as their
consumption during operations, water vapor
from evaporative loss, effluent waters with
entrained organic and inorganic materials
not otherwise suitable for recycled use
within the operation, solid biomass residue
fractions not included in algal constituent
products or indirect products, and solid
inorganic, organic, and biologic particulates
that can become airborne emissions or
suspended in liquid effluents.
A typical total lifecycle analysis will also
consider the total impact of construction,
operation and decommissioning. Gaseous
emissions and liquid and solid discharges
from commercial algal production
operations, from a life cycle analysis
perspective, include those incurred during
facilities construction prior to the initiation
of operations during the operating life of the
facility and after operations cease and the
facility is decommissioned. Emissions during
site preparation and facilities infrastructure
construction will include dust from
construction and vehicle traffic, direct and
indirect CO2 emissions from fuel combustion
for power generation and operation of
vehicles and construction equipment.
Additional CO2 , water, and other solid, liquid,
and gaseous emissions would be embedded
in the production of building materials,
equipment, fuels, and other consumables
(e.g., fertilizers and chemicals) used in
facilities construction and algal production
and processing operations. Similar emissions
of dust and CO2 will be embodied in the
decommissioning and possible restoration
of facilities sites to original condition.
Algal production operations will need
to be mindful of the
potentially applicable
regulatory requirements of
the jurisdictions in which
they are sited and obtain
the appropriate government
issued permits, licenses and
other authorizations. As part
of the process of obtaining
permits, some jurisdictions
may require decisions
regarding siting and operation
of a facility be subject to
a public review process in
order to identify the potential
impact of the facility on the
environment and to determine
what must be done to avoid
or mitigate any significant environmental
impacts. A highly generalized view of typical
major steps in the permitting process is
presented in Figure 1 to this Appendix C.
Site/Facility
Selection
Apply for
Required
Permits
Engineer
Design
Administrative
Process:
Impact
Assessment
Identify
Applicable
Regulations &
Responsible
Regulatory
Agency
Administrative
Process: Public
Participation &
Comment
By accepting government issued permits
or licenses, owners and operators of algal
production operations are also accepting
the responsibility to comply with permit
Issue
Resolution
Unresolved
Resolved
Administrative
& / or Judical
Hearings
Permit
Issued
Resolved
Favorably
Resolved
Unfavorbly
Issues
Identified
Permit
Denied
Construct or
Install Facility
Algae Strains & Farm
Inputs/Outputs
Cultivation & Production
Processes
R&D Specific Regulation
• NIH - rDNA Guidlines,
• HHS Screening Framework, DS rDNA
• EPA TSCA - TERA, R&D containeduse exemption
• USDA - GMO importation, interstate
movement and open system
research with known plant pests
R&D and Commercial
• State biotechnology and/ or
aquaculture regulations
• Federal, state and/or local permits
(air, water intake, air emissions,
wastewater, storm water discharge,
RCRA, OSHA, building and fire codes
etc.) as applicable.
R&D Specific Regulation
• EPA TSCA - TERA, R&D contained
use exemption
• Commercail Specific Regulation
• EPA TSCA - MCAN
• EPA FIFRA - pest management
• FDA - for FDA-regulated products
• TTB - for ethanol production
• NEPA, ESA may apply if federal
funding
R&D and Commercial
• USDA - plant pests in open and
closed systems
• OSHA - General duty clause
• State biotechnology and/or
aquaculture regulations
• Federal, state and/or local permits
(air, water intake, air emissions,
wastewater, storm water discharge,
RCRA, OSHA, building and fire
codes etc.) as applicable.
conditions, limitations and statutory
and regulatory requirements. In some
jurisdictions, the consequences of noncompliance may include administrative
remedies, civil court remedies, and criminal
remedies, e.g., modification of permit
conditions, permit revocation, fines and
imprisonment.
A fairly comprehensive list of U.S.
Algae-Based Products
Product Specific Regulation
• Chemicals - EPA - TSCA PMN
• Fuel - EPA /DOD/DOT/FAA fuel certification
• Fuel - EPA - RFS compliance
• Ethanol - TTB
• Food - FDA CFSAN
• Feed - FDA CVM / AAFCO
• Plus items listed as R&D Specific
environmental laws administered by the
U.S. Environmental Protection Agency can
be found at http://www.epa.gov/lawsregs/
laws/ with links to the listed laws. The
primary federal regulations for protection of
the environment are in the Title 40 Code of
Federal Regulations (CFR); however, there are
also state and local regulatory requirements
to be considered. For genetically enhanced
algae, the Coordinated Framework for
the Regulation of Biotechnology will be a
guiding document. One of the steps in the
generalized permitting process is identifying
the applicable regulatory programs and
responsible regulatory agencies. Figure 2
provides a tabular representation of some
of the agencies, regulatory programs and
guidelines (identified by acronyms) that may
be relevant to algal farm operations in the
U.S. It should be understood that parallel
regulatory programs and guidelines will be
applicable in most countries.
The ABO believes that its members will
conduct algal production operations
responsibly and in compliance with the
requirements of the nation or jurisdiction in
which their facilities are sited.
Wastewater may be used in microalgae
cultivation for applications that do not
involve human consumption of the biomass.
Instead, wastewaters are best used to makeup losses of water and nutrient at facilities
growing algae for biofuel feedstock. In
certain circumstances, algae cultivation
in wastewater can also serve to treat the
wastewater to meet regulatory discharge
limits.
The composition of wastewaters is often
complex and changing. Municipal and
agricultural wastewaters are usually rich in
carbon, nitrogen, and phosphorus, which
are primary nutrients required for algal
cultivation. However, these nutrients are
often not balanced for optimal algae growth.
Trace nutrients needed by algae may also be
present in wastewaters, and some industrial
wastewater sources would also be suitable
for algae production.
Microalgae have been grown in wastewaters
ranging from diluted swine and cattle
slurries to clear, but nitrogen-rich, subsurface
agricultural drainage. Municipal wastewater,
however, is the most studied for algae
cultivation, and typically at least some
pretreatment is performed prior to algae
cultivation. The most basic pretreatment
is screening and settling of large particles,
known as primary clarification. Algae can
also be grown on wastewater that has been
fully treated for removal of waste organic
matter, known as secondary treatment.
by discharge to receiving waters. Smaller
flows of wastewater may also originate in
algae cultivation from, for example, biomass
dewatering and processing.
In the United States, wastewater discharges
are regulated by the US Environmental
Protection Agency through the National
Pollutant Discharge Elimination System
(NPDES) permit process, with authority
delegated to state agencies. If wastewater
is diverted to algal cultivation prior to full
treatment to NPDES permit requirements,
it is highly likely that such water, after use
in algal cultivation, could only be released
under a NPDES permit. Either the water
would have to be routed back to the
permitted treatment plant for discharge
under that NPDES permit, or the algal
cultivation facility would have to operate
under a NPDES permit.
The NPDES permit system has been in
place for 40 years with many precedents,
rulings, and some revisions that are well
understood by practicing public and private
sector professionals in this field. The ABO
and the Technical Standards Committee
welcome the participation of professionals
in more fully understanding the implications
of employing wastewater nutrients in the
cultivation of algae and the implications of
the NPDES permit process.
Even absent import of wastewater to algae
farms, algae cultivation produces its own
wastewater, which may be regulated. At
algae farms where the growth media
are recycled repeatedly, salts and other
inhibitory compounds will accumulate,
leading to the need to blowdown (dispose
of ) some or all of the media periodically.
Options for blowdown management include
evaporation basins or treatment followed
Appendix E: Regulatory and Process Considerations
for Marketing Algal-based Food, Feed and Supplements
The cultivation and processing of algae for
the food and supplement market has been
heralded as an attractive market opportunity
for algae that can provide high economic
margins even at modest plant size. Algal
biomass may contain edible oils, proteins,
carbohydrates, pigments, antioxidants and
other useful dietary ingredients or additives.
Algae based food and supplement, now
mostly found in health food stores, are
expected to become increasingly common
in the mainstream food market.
The challenge confronting algae for
food producers is compliance with wellestablished but potentially complex
regulations. However, a significant number
of producers have successfully navigated
these regulations including companies such
as Cyanotech, Earthrise, Solazyme, Roquette
and others. The regulations covering this
market vary from country to country and so
only U.S. regulations are considered in this
document release.
Regulatory Framework
for Food in the U.S.
The Environmental Protection Agency
(EPA), Food and Drug Administration (FDA),
United States Department of Agriculture
(USDA), Federal Trade Commission (FTC),
Association of Animal Feed Control Officials
(AAFCO) and the International Organization
for Standardization (ISO) all have regulatory
authority over various aspects of food
production, distribution and marketing. The
FDA regulates the safety of all foods except
most meat and poultry products which
fall under the purview of the USDA. FDA
authority covers foods, dietary supplements,
food additives, color additives, medical
foods and infant formulas. EPA regulates
pesticides, including residues on food, and
antimicrobials.
In the U.S. food is defined as “articles
used for food or drink for man or other
animals, chewing gum, and articles used
for components of any such article.” 1 Algae
biomass and other products will be either
a food for human consumption, a dietary
supplement which is a subset of food, or
feed for animal consumption. The FDA
also requires that food, feed, and dietary
supplement production follow current Good
Manufacturing Practices (GMP), which is an
extensive checklist covering food production
and storage.2 The recent passage of the
Food Safety Modernization Act (FSMA)
introduced many new requirements for the
food and feed industry and FDA guidance
has not yet been published for many of
these requirements. In April 2012, the FDA
also published new guidelines on safety
assessment of food nano-materials. Some
microalgae cell and cell fragment sizes may
qualify as nano-scale materials.3 Food or
1
http://www.usp.org/food-ingredients
http://www.fda.gov/Food/GuidanceRegulation/CGMP/default.
htm
2
http://www.ift.org/food-technology/past-issues/2011/august/features/assessing-the-safety-of-nanomaterials-in-food.
aspx?page=viewall
3
Proposed
Ingredient
NDIN to FDA
NDI or GRAS
GRAS Determination
Self-determined
GRN to FDA
FDA non-response
(75 days)
FDA response
(180 days)
Table 1. Regulatory Considerations in the U.S. Food and Feed Markets.
feed companies may also require other
certifications, such as ISO 9000, which may
be necessary for international sales.4
Foods for Human Consumption
Foods can be made using algae as the main
constituent, e.g. algal flour, as an additive,
e.g. algae in energy drinks or as the source
for a supplement, e.g., capsules containing
omega-3 fatty acids. In the U.S., an algae
product intended for human consumption
falls into one of four regulatory categories:
GRAS is a food additive exemption and is a
more common approach for new foods.5 A
GRAS substance can meet current regulatory
requirements through preparation of either
a GRAS self-determination (no notice to
FDA required) or filing a GRAS notification
with the FDA. An FDA review of a GRAS
notification (GRN) requires approximately
180 days and the submission becomes
publicly accessible. Current GRNs can be
Food ingredient Market
4
www.iso.org/iso/iso_9000
http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/ucm083022.htm
5
Consumer
Feed definition
(monograph)
Feed definition
(monograph)
Consumer
acceptance
Commercial
Customers
ISO 9000
ISO 9000
ISO 9000
EPA
Pesticides/
atimicrobials
Pesticides/
atimicrobials
Pesticides/
atimicrobials
FDA
GMP, GRAS, HARPC,
Importer verification
(if applicable); NDIN
GMP, GRAS,
HARPC, NDIN
GMP, GRAS,
HARPC, NDIN
Organic standards
Organic standards
accessed on the FDA website.6 A GRAS
safety dossier contains the product’s
biological and/or chemical identity and
characterization, product specifications
and batch data, the method of production,
the intended use including food types, the
estimated dietary intakes of the product, and
a review of the publicly available scientific
literature and supporting studies. Once a
substance is determined to be GRAS, it is
GRAS only for the intended use and amounts
specified in the GRAS determination.
A dietary supplement is composed of
one or more dietary ingredients. Dietary
supplements are governed by their own set
of regulations, DSHEA7 (Dietary Supplement
Health and Education Act of 1994). New
dietary ingredients require a new dietary
ingredient notification (NDIN) to the FDA.
The term “new dietary ingredient” means a
dietary ingredient that was not marketed in
the United States in a dietary supplement
before October 15, 1994. Draft guidance for
the preparation of a NDIN is provided on the
FDA website.8 A substance that is GRAS may
be used in a dietary supplement without a
NDIN.
Foods and Feeds for Animal Consumption
Algal products intended for animal food or
feed require a GRAS notification to FDA or an
approval from the Association of American
Feed Control Officials (AAFCO) which is a
collection of state officials. States impose
Organic standards
Commercial
Customers
GRAS, NDIN,
Labeling information
& claims
Adverse
event
reporting
Health claims
Consumer
complaints
Organic standards
Consumer
complaints
regulations, inspection and license fees on
pet, specialty pet and animal foods, which
are summarized by AAFCO.9 Algae-derived
pet and animal feed additives require AAFCO
certification, often referred to as an AAFCO
monograph. Buyers must comply with
AAFCO, which is a different set of regulations
than FDA.
Processing Considerations: Growth and
Harvesting of Algae for Food
Algal growth operations producing material
intended for the food market must adhere
to a number of regulatory requirements
that are designed to assure consumer
safety. There are three key regulatory
considerations to marketing an algae food
product in the U.S. First and foremost, a
food facility that manufactures, produces,
packages, or holds food for consumption
in the U.S. must be registered with the
FDA regardless of whether it is located
in the U.S. or not.10 Second, the facility,
whether foreign or domestic, must follow
GMP regulations. And, third, the product
must have either approval as a food
additive or a determination of the GRAS
status of the ingredient. Any changes to
the manufacturing process will require
additional review.
Other considerations include setting product
specifications based on knowledge of the
algae biomass, constituent substances, and
manufacturing process to assure safety.
Food grade specifications can be found in
the current Food Chemicals Codex, (FCC),
Safety information includes pertinent
scientific information, including publicly
available scientific articles on the safety and
toxicity associated with human consumption
of the algae or extract and closely related
materials. Documentation should include
credible information on known adverse
effects associated with ingestion of algae or
extract, or closely related materials and any
available independent safety or toxicology
assessments. Algae species new to the
human food supply chain must undergo
additional toxicology tests and, in some
cases, dietary tests with animals, before
approval as a food additive or determination
of the GRAS status can be made.
Other regulatory considerations include
worker safety at the facilities and
marketing standards. The Occupational
Safety and Health Administration (OSHA)
regulates worker safety, which includes
facilities, training, clothing and accident
documentation. Compliance with organic
standards is the responsibility of the USDA
and their National Organic Standards Board
(NOSB) and claims such as “organic” require
an additional set of constraints provided by
the NOSB.13 Environmental regulations for
algal food and feeds are similar to biofuels
production.
http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.
cfm?rpt=grasListing
6
7
Dietary Supplement Market
Marketing
Feed definition
(monograph)
Abbrevations:
AAFCO=Association of Animal Feed Control Officials; EPA = Environmental Protection Agency; FDA = Food and Drug
Administration; FTC = Federal Trade Comission; GMP = Good Manufacturing Practice; GRAS = Generally Recongnized as Safe;
HRPC = Hazard Analysis and Risk-based Preventive Controls; HACCP = Hazard Analysis Critical Control Point; ISO = International
Organization for Standardization; NDIN = Dietary Ingredient Notification; DSHEA = Dietary Supplement Health and Education
Notes:
HARPC is not required for dietary supplements.
NDIN applies to new dietary ingredients for dietary supplements only.
•a generally recognized as safe (GRAS
ingredient with food additive exemption;
or,
Food ingredients are used in a variety
of products with regulatory definitions,
including conventional foods, foods for
special dietary use (medical foods), and
infant formulas. The first step in ensuring
regulatory compliance of a proposed
food additive is to determine whether
the additive should be a food additive or
a Generally Recognized as Safe (GRAS)
substance. A food additive requires that a
petition be submitted to the FDA for review
and approval, followed by a public comment
period, and then published in the Federal
Register. This can be a protracted process
that is avoided when possible.
Manufacturing
Feed definition
(monograph)
USDA
•a color additive
Note that a color additive also requires
a different regulatory process with
substantially more testing than other food
ingredients as well as FDA review and
approval. The common marketing terms
“nutraceutical,” “functional food,” and “animal
dietary supplement” do not have regulatory
recognition in the U.S.
Growing
AAFCO
FTC
•a food additive;
•a dietary supplement.
Agency or Body Raw Materials
which includes specifications for many
major chemical constituents such as ash,
moisture, and heavy metal content.11 Also,
the U.S. Pharmacopeial Convention (USP) is
a scientific nonprofit organization that sets
standards for the identity, strength, quality,
and purity of medicines, food ingredients,
and dietary supplements manufactured,
distributed and consumed worldwide.11
FSMA introduced new requirements for
food facilities including preparation of a
hazard analysis and risk-based preventive
controls (HARPC). HARPC requires food
facilities to evaluate hazards including
chemical, biological, physical, radiological,
natural toxins, pesticides, etc. that may
potentially contaminate the product. FDA
has yet to develop guidance for preparation
of a HARPC analysis. However a more
or less similar requirement, HACCP12 is
mandatory for meat, poultry, seafood and
cut vegetables even though some producers
of other foods and dietary supplement
companies obtain HACCP certifications for
added safety.
http://www.fda.gov/food/DietarySupplements/default.htm
http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/dietarysupplements/ucm257563.
htm
8
http://www.aafco.org/Portals/0/AAFCO/state_regulatory_requirement_summary_2011-2012.pdf
9
http://www.fda.gov/food/guidanceregulation/foodfacilityregistration/default.htm
10
11
http://www.usp.org/food-ingredients
12
http://www.fda.gov/food/guidanceregulation/HACCP
http://usda.gov/wps/portal/usda/usdahome?navid=ORGANIC_
CERTIFICATIO
13
Appendix F: Requirements for Marketing a Biofuel
The production of marketable bio-based
fuels from algae is an important and exciting
aspect of the algal industry. New refinery
technologies are being developed to
construct these fuels from algal biomass,
extracted oils and volatiles like alcohol that
are generated by algae. However, new fuels
must meet a complex set of regulatory and
commercial requirements before they can
be marketed. These include environmental
regulations, safety and infrastructure
compatibility, and engine compatibility.
In the United States the Clean Air Act
prohibits sale of gasoline or diesel fuel that
is not “substantially similar” to conventional
fuel; and defines this as not causing or
contributing to the degradation of a vehicle’
emission control system. In general these
fuels are hydrocarbons meeting their
respective ASTM standards, however EPA
has ruled that aliphatic alcohols (except
methanol) and ethers can be blended in
gasoline at up to 2.7 wt% oxygen and meet
this requirement. Aliphatic alcohols can
Testing and Labeling
Food safety and security regulations
require constant culture monitoring and
documentation for the possible presence
of toxins, bacteria, heavy metals, chemical
residuals and other contaminants. GMP
requires records from each batch of food
quality control tests (21 CFR Part 110).
The FDA’s Food Labeling Guide and a
series of FDA guidance documents detail
food labeling regulations including
nutritional facts and ingredient labeling
on packaged products.14 Labels that
make health claims require additional
documentation be provided to FDA for
proof. Dietary supplement labeling must
comply with specific FDA regulations (21
CFR Part 111).15 For example, New York state
Attorney General Eric Schneiderman issued
subpoenas in August 2012 for 5-Hour Energy
and Monster drinks that had promised
healthy bursts of energy.
Conclusion
Food regulations vary depending on the
intended use and market as well as country.
http://www.fda.gov/food/ingredientspackaginglabeling/labelingnutrition/ucm2006860.htm
14
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
cfrsearch.cfm?cfrpart=111
15
literature search and detailed engine
emissions speciation study. To date this has
been completed for ethanol (in gasoline)
and biodiesel.
EPA also regulates underground storage
tanks to protect ground water and requires
that these tanks must be compatible with
the materials stored in them. Compatibility
can be demonstrated by third party testing
(such as Underwriters Laboratories, UL) or
by the manufacturer of the tank providing
warranty coverage for use with the new
fuel. Above ground equipment such as fuel
dispensers, hoses and nozzles is required
to have a third party listing as being
compatible with the fuel being handled by
both the Occupational, Safety, and Health
Administration and typically also by local
fire marshals. Third party testing normally
requires that the fuel have an ASTM standard
to serve as the basis for UL to develop a test
fluid, and that the manufacturers will be
willing to submit their equipment for testing
and potential listing by UL.
requirements. Individual states are
responsible for regulating fuel quality as part
of consumer protection laws, and a majority
of states use ASTM standards for this
purpose. Producers of an ethanol, fatty acid
methyl ester, or hydrocarbon biofuel may be
able to demonstrate that it meets existing
ASTM standards. In some cases a blendstock
standard may be required, such as D4806 for
denatured fuel ethanol or D6751 for B100
biodiesel intended for blending at up to 20
vol%.
While meeting all of these requirements
requires a major effort, there have been
a number of new fuels approved by EPA
recently. In particular Dynamic Fuels, 1
Solazyme, 2 and KiOR 3 have successfully
registered renewable hydrocarbon fuels.
Gevo has received EPA registration for its
isobutanol product 4 while competitor
Butamax anticipates approval in 2014. 5 The
ASTM Petroleum Products Committee has
approved a fuel butanol standard that will
be published later in 2013.
In the U.S., several federal as well as state
agencies may have authority over the
production and marketing of a foods, feeds,
and dietary supplements. Algae producers
should review the pathways to market and
be aware of the requirements. Poor planning
could result in lost time and money, and
potentially, legal action.
also be blended into gasoline at 3.7% wt%
oxygen under the Octamix waiver, that
requires the inclusion of specific corrosion
inhibitor additives. Other materials must
demonstrate that they will not cause or
contribute to the degradation of vehicle
emission control systems and can apply
for a waiver of the substantially similar
requirement. There are no corresponding
substantially similar rulings for diesel fuel,
but the same concepts apply. A second
EPA requirement is fuel registration, which
for biomass-derived materials regardless
of composition will require a health effects
The fuels market is a commodity market.
Products from different manufacturers are
fungible, interchangeable as long as they
meet a common ASTM standard. ASTM
standards are developed by consensus of
ASTM members (anyone can join ASTM).
Members include fuel producers and
distributors, engine and car makers, state
fuel regulators, and other interested parties.
ASTM standards are typically focused on
ensuring safety in distribution and handling,
as well as fuel-engine compatibility. ASTM
standards may also be used to describe fuels
for purposes of meeting EPA fuel registration
http://www.globenewswire.com/newsroom/news.
html?d=237679
1
http://www.algaeindustrymagazine.com/solazyme-receives-fuelregistration/?utm_source=feedburner&utm_medium=email&utm_
campaign=Feed%3A+AlgaeIndustryMagazine+%28Alg
ae+Industry+Magazine%29
2
3
http://investor.kior.com/releasedetail.cfm?ReleaseID=694743
http://eponline.com/articles/2010/11/13/gevos-isobutanolsecures-epa-registration-for-use-as-fuel-additive.aspx
4
Kris, Bill “Can biobutanol provide ethanol producers new paths
towards diversification?” Ethanol Producer Magazine, March 2012.
5
Appendix G: Quality Attributes and Considerations for Trading Algal Oils.
purity (crude, refined, refined and bleached)
or their end use (technical grade, feed
grade, etc.). The majority of oils and fats
are marketed into the food industry as
shortenings, salad oils, margarine, frying oils,
or as components in baking, mayonnaise
or salad oils. These oils put a premium on
properties important to their end use (color,
cold properties, heat stress properties),
minor compounds that can effect flavor or
texture (impurities, unsaponifiable material),
other properties that can affect shelf life
(moisture, storage stability), and overall
purity of the triglyceride oil (i.e. low level of
free fatty acids).
Some of these same oils/fats are marketed
as building blocks for detergents, lubricants
or other industrial or cosmetic applications.
These markets capitalize on the properties
that are already present from its other
Algal oils have the potential to be marketed
and sold into both existing markets for oils
and fats—such as cooking oils or feedstocks
for biodiesel—as well as newly emerging
markets such as aviation fuels, biocrudes,
bioplastics, and biopolymers. Most of these
applications utilize the oil in its entirety
but there is also interest in high value
applications for specialty oils or for specialty
oil components, such as omega-3 or
omega-6 fatty acids used in nutraceuticals.
Trading rules and important quality
attributes of existing oils and fats are
well established. They are based on a
combination of key attributes common to
naturally occurring triglycerides (oils/fats)
or important characteristics of the use of
the oil/fat. The algal industry will need to
provide characteristics of algal oil allowing
it to be easily compared to existing oils/fats.
This will facilitate its acceptance into both
existing and emerging markets as well as
simplify buyer/seller agreements, hedging,
and other aspects important to the buying/
selling of oils and fats. While it is premature
at this time to develop specific standards or
trading rules of algal oils, those for existing
oils/fats can serve as a useful guide for the
attributes and specific values of algal oils
that will be demanded by customers.
Traditional oils and fats are generally broken
down first by whether they are edible or
inedible, by their source (soybean, corn,
beef, pork, sunflower, used cooking oil,
etc.), and then by various industry terms
which generally describe their quality or
uses, and typically add or emphasize one
or several properties that are important
for the particular application. Fatty acid
chain length is particularly important in
the detergent markets, with shorter chain
lengths such as those found in coconut oil
are important for some applications while
longer chain lengths such as beef tallow or
are important for others.
Inedible oils—which include used cooking
oils, most animal fats, and some inedible
vegetable oils such as rapeseed oil or castor
oil—are commonly used for animal feed
rations, biodiesel, and other industrial
applications. These applications tend to be
less concerned with color, some impurities,
and free fatty acid level as the processed
used are better able to deal with a lower
grade oil/fat. Some other properties
become important for these applications
such as viscosity, BTU content, and low levels
of poly-unsaturated fatty acids to enhance
storage stability.
Various trade groups publish trading
standards, guidelines, or quality standards
for oils and fats.1,2,3,4 A summary of the most
common quality characteristics, testing
National Oilseed Processors Association, Trading Rules for the
Purchase and Sale of Soybean Oil, www.nopa.org
1
methodologies, and values are incorporated
into the table below. Different end users or
customers may want additional information
(i.e. more specific fatty acid profile for high
value nutraceuticals), but the attributes and
values below provide some good targets for
the types of information that will need to be
provided in order for the algal oil industry to
continue to grow. Algal oil interests should
carefully consider these existing markets and
their requirements—and how algal oils may
provide advantages compared to existing
oils/fats—as the industry develops.
AOCS (American Oil Chemist Society), Ck 1-07 Analytical Guidelines for Assessing Feedstock to Ensure Biodiesel Quality, www.
aocs.org
2
National Renderers Association, Pocket Information Manual: A
Buyers Guide to Rendered Products, www.renderers.org
3
National Institute of Oilseed Products, Trading Rules (covers
vegetable oils other than soybean oil), www.niop.org
4
Members | 2013
Platinum
Gold
Corporate
Algae Industry Incubation Consortium Japan
AMEC
Aurora Algae, LLC
Battelle Pacific Northwest Division
Cereplast
Church & Dwight
Colorado Lining International
Combined Power Cooperrative
Diversified Technologies, Inc.
Earthrise Nutritionals, LLC
ECO2Capture
Electric Power Research Institute
Evodos B.V.
FedEx Express
Fredrikson & Byron, P.A.
GF Piping Systems
Greenfield Ethanol, Inc.
International Air Transport Association
IGV Institut fuer Getreideverarbeitung GmbH
Keller and Heckman LLP
Kimberly-Clark Corporation
MicroBio Engineering, Inc.
MTU Aero Engines GmbH
Muradel Pty Ltd
National Alliance for Advanced Biofuels and Bio-Products
National Center for Marine Algae
and Microbiota
Neste Oil Corporation
OpenAlgae
OriginOil, Inc.
Petrixo
Phycal, LLC
POS Bio-Sciences
Saudi Basic Industries Corporation
SCHOTT North America
Solix Biofuels, Inc.
Solutions 4CO2, Inc.
Stoel Rives, LLP
Subitec GmbH
Synthetic Genomics
Texas AgriLife Research
The Linde Group
The Scoular Company
European Algae Biomass Association
National Biodiesel Board
Phycological Society of America
Supporting Organizations
American Oil Chemists’ Society
Biotechnology Industry Organization
National Laboratories
Los Alamos National Laboratory
National Renewable Energy Laboratory
Pacific Northwest National Laboratory
Sandia National Laboratory

Industrial Algae Measurements

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